Crystalline form of (s)-n-(5-((r)-2-(2,5-difluorophenyl)-pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide hydrogen sulfate

ABSTRACT

A novel crystalline form of (S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide, pharmaceutical compositions containing said crystalline form and the use of said crystalline form in the treatment of pain, cancer, inflammation, neurodegenerative disease or  Trypanosoma cruzi  infection are disclosed. In some embodiments, the novel crystalline form comprises a stable polymorph of (S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide hydrogen sulfate. The present invention is further directed to a process for the preparation of the novel crystalline form.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.Nos. 62/080,374, filed Nov. 16, 2014, and 62/169,545, filed Jun. 1,2015, both of which are incorporated in their entireties herein.

BACKGROUND

1. Field of the Invention

The present disclosure relates to(S)—N-(5-((R)-2-(2,5-difluorophenyl)-pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide(Formula I) and to pharmaceutically acceptable salts thereof, forexample the hydrogen sulfate salt, and further to a novel crystallineform of the hydrogen sulfate salt, which exhibit Trk family proteintyrosine kinase inhibition, pharmaceutical compositions containing thesame, processes of making the crystalline form, and the use of thecompound and crystalline form in the treatment of pain, inflammation,cancer, and certain infectious diseases.

2. Description of the Related Art

Trk's are the high affinity receptor tyrosine kinases activated by agroup of soluble growth factors called neurotrophins (NT). The Trkreceptor family has three members—TrkA, TrkB and TrkC. Among theneurotrophins are (i) nerve growth factor (NGF) which activates TrkA,(ii) brain-derived neurotrophic factor (BDNF) and NT-4/5 which activateTrkB and (iii) NT3 which activates TrkC. Trk's are widely expressed inneuronal tissue and are implicated in the maintenance, signaling andsurvival of neuronal cells (Patapoutian, A. et al., Current Opinion inNeurobiology, 2001, 11, 272-280).

Recent literature has shown that overexpression, activation,amplification and/or mutation of Trk's are associated with many cancersincluding neuroblastoma (Brodeur, G. M., Nat. Rev. Cancer 2003, 3,203-216), ovarian cancer (Davidson., B. et al., Clin. Cancer Res. 2003,9, 2248-2259), breast cancer (Kruettgen et al., Brain Pathology 2006,16: 304-310), prostate cancer (Dionne et al., Clin. Cancer Res. 1998,4(8): 1887-1898), pancreatic cancer (Dang et al., Journal ofGastroenterology and Hepatology 2006, 21(5): 850-858), multiple myeloma(Hu et al., Cancer Genetics and Cytogenetics 2007, 178: 1-10),astrocytoma and medulloblastoma (Kruettgen et al., Brain Pathology 2006,16: 304-310), glioma (Hansen et al., Journal of Neurochemistry 2007,103: 259-275), melanoma²⁵, thyroid carcinoma (Brzezianska et al.,Neuroendocrinology Letters 2007, 28(3), 221-229), lung adenocarcinoma(Perez-Pinera et al., Molecular and Cellular Biochemistry 2007,295(1&2), 19-26), large cell neuroendocrine tumors¹⁹ (Marchetti et al.,Human Mutation 2008, 29(5), 609-616), and colorectal cancer (Bardelli,A., Science 2003, 300, 949). In preclinical models of cancer, Trkinhibitors are efficacious in both inhibiting tumor growth and stoppingtumor metastasis. In particular, non-selective small molecule inhibitorsof TrkA, TrkB, TrkC and Trk/Fc chimeras were efficacious in bothinhibiting tumor growth and stopping tumor metastasis²⁵ (Nakagawara, A.(2001) Cancer Letters 169:107-114; Meyer, J. et al. (2007) Leukemia,1-10; Pierottia, M. A. and Greco A., (2006) Cancer Letters 232:90-98;Eric Adriaenssens, E. et al. Cancer Res (2008) 68:(2) 346-351).Therefore, an inhibitor of the Trk family of kinases is expected to haveutility in the treatment of cancer.

In addition, inhibitors of the Trk/neurotrophin pathway have beendemonstrated to be effective in numerous pre-clinical animal models ofpain. For example, antagonistic NGF and TrkA antibodies (for example,RN-624) have been shown to be efficacious in inflammatory andneuropathic pain animal models and in human clinical trials (Woolf, C.J. et al. (1994) Neuroscience 62,327-331; Zahn, P. K. et al. (2004) J.Pain 5, 157-163; McMahon, S. B. et al., (1995) Nat. Med. 1, 774-780; Ma,Q. P. and Woolf, C. J. (1997) Neuroreport 8, 807-810; Shelton, D. L. etal. (2005) Pain 116, 8-16; Delafoy, L. et al. (2003) Pain 105, 489-497;Lamb, K. et al. (2003) Neurogastroenterol. Motil. 15, 355-361; Jaggar,S. I. et al. (1999) Br. J. Anaesth. 83, 442-448). Additionally, recentliterature indicates after inflammation, BDNF levels and TrkB signalingis increased in the dorsal root ganglion (Cho, L. et al. Brain Research1997, 749, 358) and several studies have shown antibodies that decreasesignaling through the BDNF/TrkB pathway inhibit neuronalhypersensitization and the associated pain (Chang-Qi, L et al. MolecularPain 2008, 4:27).

It has been shown that NGF secreted by tumor cells and tumor invadingmacrophages directly stimulates TrkA located on peripheral pain fibers.Using various tumor models in both mice and rats it was demonstratedthat neutralizing NGF with a monoclonal antibody inhibits cancer relatedpain to a degree similar or superior to the highest tolerated dose ofmorphine. In addition, activation of the BDNF/TrkB pathway has beenimplicated in numerous studies as a modulator of various types of painincluding inflammatory pain (Matayoshi, S., J. Physiol. 2005,569:685-95), neuropathic pain (Thompson, S. W., Proc. Natl. Acad. Sci.USA 1999, 96:7714-18) and surgical pain (Li, C.-Q. et al., MolecularPain, 2008, 4(28), 1-11). Because TrkA and TrkB kinases may serve as amediator of NGF driven biological responses, inhibitors of TrkA and/orother Trk kinases may provide an effective treatment for chronic painstates.

The current treatment regimes for pain conditions utilize severalclasses of compounds. The opioids (such as morphine) have severaldrawbacks including emetic, constipatory and negative respiratoryeffects, as well as the potential for addictions. Non-steroidalanti-inflammatory analgesics (NSAIDs, such as COX-1 or COX-2 types) alsohave drawbacks including insufficient efficacy in treating severe pain.In addition, COX-1 inhibitors can cause ulcers of the mucosa.Accordingly, there is a continuing need for new and more effectivetreatments for the relief of pain, especially chronic pain.

In addition, inhibition of the neurotrophin/Trk pathway has been shownto be effective in treatment of pre-clinical models of inflammatorydiseases. For example, inhibition of the neurotrophin/Trk pathway hasbeen implicated in preclinical models of inflammatory lung diseasesincluding asthma (Freund-Michel, V; Frossard, N.; Pharmacology &Therapeutics (2008), 117(1), 52-76), interstitial cystitis (Hu Vivian Y;et. al. The Journal of Urology (2005), 173(3), 1016-21), inflammatorybowel diseases including ulcerative colitis and Crohn's disease (DiMola, F. F., et. al., Gut (2000), 46(5), 670-678) and inflammatory skindiseases such as atopic dermatitis (Dou, Y.-C.; et. al. Archives ofDermatological Research (2006), 298(1), 31-37), eczema and psoriasis(Raychaudhuri, S. P.; et. al. Journal of Investigative Dermatology(2004), 122(3), 812-819).

The neurotrophin/Trk pathway, particularly BDNF/TrkB, has also beenimplicated in the etiology of neurodegenerative diseases includingmultiple sclerosis, Parkinson's disease and Alzheimer's disease(Sohrabji, Farida; Lewis, Danielle K. Frontiers in Neuroendocrinology(2006), 27(4), 404-414). Modulation of the neutrophin/Trk pathway mayhave utility in treatment of these and related diseases.

The TrkA receptor is also thought to be critical to the disease processin the infection of the parasitic infection of Trypanosoma cruzi (Chagasdisease) in human hosts (de Melo-Jorge, M. et al. Cell Host & Microbe(2007), 1(4), 251-261). Thus, TrkA inhibition may have utility intreating Chagas disease and related protozoan infections.

Trk inhibitors may also find use in treating disease related to animbalance of the regulation of bone remodeling, such as osteoporosis,rheumatoid arthritis, and bone metastases. Bone metastases are afrequent complication of cancer, occurring in up to 70 percent ofpatients with advanced breast or prostate cancer(1) and in approximately15 to 30 percent of patients with carcinoma of the lung, colon, stomach,bladder, uterus, rectum, thyroid, or kidney. Osteolytic metastases cancause severe pain, pathologic fractures, life-threatening hypercalcemia,spinal cord compression, and other nerve-compression syndromes. Forthese reasons, bone metastasis is a serious and costly complication ofcancer. Therefore, agents that can induce apoptosis of proliferatingosteoblasts would be highly advantageous. Expression of TrkA and TrkCreceptors has been observed in the bone forming area in mouse models ofbone fracture (K. Asaumi, et al., Bone (2000) 26(6) 625-633). Inaddition, localization of NGF was observed in almost all bone formingcells (K. Asaumi, et al.). Recently, it was demonstrated that a pan-Trkinhibitor inhibits the tyrosine signaling activated by neurotrophinsbinding to all three of the Trk receptors in human hFOB osteoblasts (J.Pinski, et al., (2002) 62, 986-989). These data support the rationalefor the use of Trk inhibitors for the treatment of bone remodelingdiseases, such as bone metastases in cancer patients.

Several classes of small molecule inhibitors of Trk kinases said to beuseful for treating pain or cancer are known (Expert Opin. Ther. Patents(2009) 19(3)).

International Patent Application Publications WO 2006/115452 and WO2006/087538 describe several classes of small molecules said to beinhibitors of Trk kinases which could be useful for treating pain orcancer.

Pyrazolo[1,5-a]pyrimidine compounds are known. For example,International Patent Application Publication WO 2008/037477 disclosespyrazolo[1,5-a]pyrimidine compounds bearing an alkyl, aryl orheterocyclic group at the 3-position. These compounds are asserted to bePI3K and/or mTOR Lipid Kinase inhibitors.

PCT Patent Publication No. WO 2008/058126 disclosespyrazolo[1,5-a]pyrimidine compounds bearing a phenyl group at the3-position. These compounds are asserted to be Pim-kinase inhibitors.

U.S. Patent Publication No. 2006/0094699 disclosespyrazolo[1,5-a]pyrimidine compounds bearing a —C(═O)NH-phenyl,—C(═O)(4-methylpiperidinyl) or —C(═O)NMe(CH₂-trimethylpyrazolyl) groupat the 3-position for use in combination therapy with a glucocorticoidreceptor agonist.

PCT Patent Publication Nos. WO 2010/033941, WO 2010/048314, WO2011/006074, and WO 2011/146336 disclose compounds which exhibit Trkfamily protein tyrosine kinase inhibition, and which are useful in thetreatment of pain, cancer, inflammation, neurodegenerative diseases andcertain infectious diseases. WO 2010/048314 discloses in Example 14A ahydrogen sulfate salt of(S)—N-(5((R)-2-(2,5-difluorophenyl)-pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide.WO 2010/048314 does not disclose the particular form of the hydrogensulfate salt described herein when prepared according to the method ofExample 14A in that document. In particular, WO 2010/048314 does notdisclose crystalline form (I-HS) as described below.

All documents, including scientific articles, patent publications andapplications, and the like, referenced in the present disclosure arehereby incorporated by reference in their entirety.

SUMMARY

The present disclosure relates to(S)—N-(5((R)-2-(2,5-difluorophenyl)-pyrrolidin-1-1)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide(Formula I) and to pharmaceutically acceptable salts thereof, forexample the hydrogen sulfate salt, and further to a novel crystallineform of the hydrogen sulfate salt, which exhibit Trk family proteintyrosine kinase inhibition, pharmaceutical compositions containing thesame, processes of making the crystalline form, and the use of thecompound and crystalline form in the treatment of pain, inflammation,cancer, and certain infectious diseases.

Provided herein is a novel crystalline form of the compound of FormulaI:

also known as(S)—N-(5-((R)-2-(2,5-difluorophenyl)-pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide.In particular, the novel crystalline form comprises the hydrogen sulfatesalt of the compound of Formula I in a stable polymorph form,hereinafter referred to as crystalline form (I-HS) and LOXO-101, whichcan be characterized, for example, by its X-ray diffraction pattern—thecrystalline form (I-HS) having the formula:

In some embodiments, crystalline form (I-HS) is characterized by havingXRPD diffraction peaks (2θ degrees) at 18.4±0.2, 20.7±0.2, 23.1±0.2, and24.0±0.2. In some embodiments, crystalline form (I-HS) is characterizedby having XRPD diffraction peaks (20 degrees) at 10.7±0.2, 18.4±0.2,20.7±0.2, 23.1±0.2, and 24.0±0.2. In some embodiments, crystalline form(I-HS) is characterized by having XRPD diffraction peaks (2θ degrees) at10.7±0.2, 18.4±0.2, 19.2±0.2, 20.2±0.2, 20.7±0.2, 21.5±0.2, 23.1±0.2,and 24.0±0.2. In some embodiments, crystalline form (I-HS) ischaracterized by having XRPD diffraction peaks (20 degrees) at 10.7±0.2,15.3±0.2, 16.5±0.2, 18.4±0.2, 19.2±0.2, 19.9±0.2, 20.2±0.2, 20.7±0.2,21.5±0.2, 22.1±0.2, 23.1±0.2, 24.0±0.2. 24.4±0.2, 25.6±0.2, 26.5±0.2,27.6±0.2, 28.2±0.2, 28.7±0.2, 30.8±0.2, and 38.5±0.2.

In some embodiments, the crystalline form (I-HS) has XRPD patternsubstantially as shown in FIG. 29.

In some embodiments, the crystalline form exhibits an onset to maximumof about 193 to about 205° Celsius, as measured by differential scanningcalorimetry. In some embodiments, the crystalline form (I-HS) exhibits aheat of melting of about 2.415 mW, as measured by differential scanningcalorimetry. In some embodiments, the crystalline form (I-HS) has a DSCthermogram substantially as shown in FIG. 26. In some embodiments, thecrystalline form (I-HS) is non-hygroscopic.

Some embodiments include a pharmaceutical composition comprising apharmaceutically acceptable carrier and crystalline form (I-HS). Someembodiments include a pharmaceutical composition made by mixingcrystalline form (I-HS) and a pharmaceutically acceptable carrier. Someembodiments include a process of making a pharmaceutical compositioncomprising mixing crystalline form (I-HS) and a pharmaceuticallyacceptable carrier.

The present disclosure also relates to methods for the treatment ofcancer, pain, inflammation, and certain infectious diseases comprisingadministering to a subject in need thereof a therapeutically effectiveamount of crystalline form (I-HS). Some embodiments include the use ofcrystalline form (I-HS) in the preparation of a medicament for treatingcancer, pain, inflammation, and certain infectious diseases, in asubject in need thereof.

Also provided herein is a method of treating a cancer mediated by a Trkkinase in a subject in need thereof, the method comprising administeringto the subject a therapeutically effective amount of crystalline form(I-HS). In some embodiments, the cancer is mediated by Trk; TrkB; orTrkA and TrkB. In some embodiments, a patient is diagnosed or identifiedas having a Trk-associated cancer.

Further provided herein is a method for treating cancer in a subject inneed thereof, the method comprising: (a) determining if the cancer isassociated with one or more of overexpression, activation,amplification, and mutation of a Trk kinase; and (b) if the cancer isdetermined to be associated with one or more of overexpression,activation, amplification, and mutation of a Trk kinase, administeringto the subject a therapeutically effective amount of crystalline form(I-HS). In some embodiments, a method for treating cancer in a subjectin need thereof is provided, the method comprising: (a) determining ifthe cancer is mediated by a Trk kinase; and (b) if the cancer isdetermined to be mediated by a Trk kinase, administering to the subjecta therapeutically effective amount of crystalline form (I-HS). Alsoprovided herein is a method of treating a subject comprising: (a)performing an assay on a sample obtained from the subject to determinewhether the subject has dysregulation of a NTRK gene, a Trk protein, orexpression or level of the same; and (b) administering to a subjectdetermined to have dysregulation of a NTRK gene, a Trk protein, orexpression or activity, or level of the same a therapeutically effectiveamount of crystalline form (I-HS).

In some embodiments, the dysregulation of a NTRK gene, a Trk protein, orexpression or level of the same is a chromosome translation that resultsin the translation of a Trk fusion protein. For example, the Trk fusionprotein is selected from the group consisting of: TP53-TrkA, LMNA-TrkA,CD74-TrkA, TFG-TrkA, TPM3-TrkA, NFASC-TrkA, BCAN-TrkA, MPRIP-TrkA,TPR-TrkA, RFWD2-TrkA, IRF2BP2-TrkA, SQSTM1-TrkA, SSBP2-TrkA,RABGAP1L-TrkA, C18ORF8-TrkA, RNF213-TrkA, TBC1D22A-TrkA, C200RF112-TrkA,DNER-TrkA, ARHGEF2-TrkA, CHTOP-TrkA, PPL-TrkA, PLEKHA6-TrkA, PEAR1-TrkA,MRPL24-TrkA, MDM4-TrkA, LRRC71-TrkA, GRIPAP1-TrkA, EPS15-TrkA,DYNC2H1-TrkA, CEL-TrkA, EPHB2-TrkA, TGF-TrkA, NACC2-TrkB, QKI-TrkB,AFAP1-TrkB, PAN3-TrkB, SQSTM1-TrkB, TRIM24-TrkB, VCL-TrkB, AGBL4-TrkB,DAB2IP-TrkB, ETV6-TrkC, BTBD1-TrkC, LYN-TrkC, RBPMS-TrkC, EML4-TrkC,HOMER2-TrkC, TFG-TrkC, FAT1-TrkC, and TEL-TrkC.

In some embodiments, the dyregulation of a NTRK gene, a Trk protein, orexpression or activity of the same is one or more point mutation in thegene. For example, the NTRK gene is a NTRK1 gene, and the one or morepoint mutations in the NTRK1 gene results in the translation of a TrkAprotein having substitutions are one or more of the following amino acidpositions: 33, 336, 337, 324, 420, 444, 517, 538, 649, 682, 683, 702,and 1879. In some embodiments, the one or more point mutations in theNTRK1 gene results in the translation of a TrkA protein having one ormore of the following amino acid substitutions: R33W, A336E, A337T,R324Q, R324W, V420M, R444Q, R444W, G517R, G517V, K538A, R649W, R649L,R682S, V683G, R702C, and C1879T.

The features and advantages described in this summary and the followingdetailed description are not all-inclusive. Many additional features andadvantages will be apparent to one of ordinary skill in the art in viewof the drawings, specification, and claims hereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an X-ray powder diffraction (XRPD) pattern ofcrystalline form (I-HS) prepared according to Example 2, according toone embodiment.

FIG. 2 illustrates a simultaneous thermogravimetric/differential thermalanalyzer (TG/DTA) profile of crystalline form (I-HS) prepared accordingto Example 2, according to one embodiment.

FIG. 3 illustrates a differential scanning calorimetry (DSC) profile ofcrystalline form (I-HS) prepared according to Example 2, according toone embodiment.

FIGS. 4A and 4B illustrate polarized light microscopy (PLM) images ofcrystalline form (I-HS) prepared according to Example 2 under (A)unpolarized and (B) polarized light, according to some embodiments.

FIG. 5 illustrates a dynamic vapor sorption (DVS) isotherm profile ofcrystalline form (I-HS) prepared according to Example 2, according toone embodiment.

FIG. 6 illustrates an infrared (IR) spectroscopy profile of crystallineform (I-HS) prepared according to Example 2, according to oneembodiment.

FIG. 7 illustrates an XRPD pattern of the amorphous freebase form of acompound of Formula I, according to one embodiment.

FIG. 8 is a graph showing the dose dependent inhibition of theproliferation of CUTO-3F lung adenocarcinoma cells harboring aMPRIP-NTRK1 fusion protein using the crystalline form (I-HS).

FIG. 9 is a graph showing the dose dependent inhibition of theproliferation of KM12 colorectal cancer cells harboring a TPM3-NTRK1fusion protein using the crystalline form (I-HS).

FIG. 10 is a graph showing the dose dependent inhibition of theproliferation of MO-91 acute myeloid leukemia cells harboring aETV6-NTRK3 fusion protein using the crystalline form (I-HS).

FIG. 11 is an immunoblot showing that the crystalline form (I-HS)inhibits the activation of MPRIP-TRKA kinase, ERK1/2 in CUTO-3F cells,and AKT activity in KM12 cells. The cells were treated for 2 hours withthe crystalline form (I-HS) at the indicated doses.

FIG. 12 is an immunoblot showing that the crystalline form (I-HS)inhibits the activation of TPM3-TRKA kinase and downstream ERK1/2 andAKT activity in KM12 cells. The cells were treated for 2 hours with thecrystalline form (I-HS) at the indicated doses.

FIG. 13 is an immunoblot showing that the crystalline form(I-HS)inhibits TEL-TRKC kinase and ERK1/2 and AKT activity in MO-91cells. The cells were treated for 2 hours with the crystalline form(I-HS) at the indicated doses.

FIG. 14 is a schematic depicting the LMNA-NTRK1 gene fusion identifiedin the patient's tumor sample: the joining of the first two exons ofLMNA (NM_170707) with exon 11-17 of NTRK1 (NM_002529).

FIG. 15 is a fluorescence micrograph from the NTRK1 break-apart FISHassay, which shows both paired green (5′ NTRK1) and red (3′ NTRK1)signals corresponding to the normal gene (yellow arrow), and isolatedred signals (red arrows) are observed in tumor nuclei (stained blue withDAPI) indicate a chromosomal deletion that leads to a NTRK1 gene fusion.

FIG. 16 is a chromatograph of DNA sequencing of the RT-PCR product usingLMNA (5′) and NTRK1 (3′) primers indicating the fusion breakpointbetween exon 2 LMNA and exon 11 of NTRK1.

FIG. 17 is a schematic of the TRK-SHC1 proximity ligation assay (PLA).This cartoon demonstrates the detection of proximal (<40 nM) TRK andSHC1 proteins in tumor cells. The TRK antibody (rabbit) used can detectthe c-terminus of TRKA (encoded by NTRK1), TRKB (NTRK2), or TRKC (NTRK3)proteins. SHC1 is detected by a SHC1 antibody (mouse). Binding ofspecies-specific secondary antibodies with covalently attachedcomplementary nucleotide sequences allows an in situ PCR reaction togenerate DNA, which can be detected by fluorescence in situhybridization visualized in the method as red dots. The assay has thepotential to detect activated TRK regardless of mechanism of activation(gene fusion, mutation, or autocrine/paracrine activation of thewildtype) of TRK receptor family member (TRKA/B/C).

FIG. 18 is a set of data that validate the TRK-SHC1 PLA. (A) The CUTO-3cell line, derived from a malignant pleural effusion from a patient withstage IV lung adenocarcinoma harboring the MPRIP-NTRK1 gene fusion, wastransfected with a non-targeting control (NTC) siRNA, NTRK1-directedsiRNA, or untreated (control) and assayed for TRKA protein expression.Western blot analysis demonstrates a marked decrease in the TRKA proteinlevels, and corresponds to the MPRIP-TRKA fusion protein that migrateswith an apparent molecular weight of 170 kD. TRK-SHC1 PLA was performedin cells treated as in (A) demonstrating a robust positive signal in thesiRNA control (B), but proportional decrease in the NTRK1 siRNA (C).CUTO-3 cells were treated with DMSO (D) or crystalline form (I-HS) at aconcentration of 100 nM (E) for 2 hours demonstrating disruption ofTRKA-SHC1 complexes in the crystalline form (I-HS) treated samplecompared to control. CULC001 is a patient-derived tumor xenograft (PDX)derived from the same tumor as the CUTO-3 cell line and harbors theMPRIP-NTRK1 gene fusion (not shown). CULC002 is a PDX from a NSCLCpatient without a known driver (ALK, ROS1, EGFR, KRAS, and BRAFnegative) and is negative for an NTRK1 gene fusion by NTRK1 break-apartFISH (not shown). TRK PLA analysis demonstrates a robust signal inCULC001 (F) but no signal in CULC002 (G) tumor nuclei. Panels (H) and(I) show a nerve bundle from the CULC001 PDX. TRK-SHC1 PLA is positiveonly in this region of the CULC002 tumor sample and is suggestive ofautocrine signaling in a TRKA, TRKB, or TRKC receptor as this family isexpressed in nervous tissue and serves as internal positive control forthis otherwise negative tumor sample.

FIG. 19 is an image from a TRK SHC1 proximity ligation assay and acontrol. (A) The TRK-SHC1 proximity ligation assay demonstrates robustsignaling in the tumor nuclei but weak signaling in the thick walledblood vessel. Nuclei were stained with DAPI (blue) and the red signalsrepresent a positive PLA indicative of TRKA-SHC1 protein complexes. Ablood vessel is indicated within the partial ellipse (dotted whiteline). (B) Adjacent tumor tissue section stained with hematoxylin andeosin indicating a thick-walled blood vessel (within partial ellipseindicated by dotted white line) and flanking tumor nuclei.

FIG. 20 are a set of images showing the TRK and ALK PLA in an ALK+tumorsample. FFPE tumor sample from an ALK+patient (autopsy sample) wasassayed using the TRK-SHC1 PLA (A) demonstrating an absence of signal orALK-GRB2 PLA (B) showing robust ALK signaling.

FIG. 21 is a set of three computed tomography images from a subjecthaving undifferentiated sarcoma. CT images were obtained followingpre-operative chemotherapy and primary tumor resection with arrowindicating the presence of an 18-mm right lung nodule (A), baselineimaging just prior to dosing with the crystalline form (I-HS) on study(B), and following 1 cycle (28 days) of dosing of with the crystallineform (I-HS) (C). The patient was observed to have metastatic diseaseonly in the lungs and therefore the CT scan images show axial (top) andcoronal (bottom) images focusing on the thoracic cavity. The imagesdemonstrate an initial rapid disease progression (A-B, 13 week interval)followed by a marked tumor response with decreased size and/orresolution of the numerous pulmonary metastases (B-C, 4 week interval).

FIG. 22 is a graph showing the serum CA125 levels in a patient havingundifferentiated sarcoma treated with crystalline form (I-HS) over time.Serum CA125 levels were found to be elevated in this patient, andsubsequently followed as a potential indicator of activity. Serum CA125was drawn at baseline (day −8) prior to dosing and at the indicated timepoint points following the initiation of dosing at day −3 through day 56demonstrating a time-dependent decrease in this tumor marker. The dashedred line indicates the upper limit of normal (35 U/mL) of thislaboratory test.

FIG. 23 is a graph showing the dose dependent inhibition of theproliferation of HCC78 cells harboring a SLC34A2-ROS1 fusion proteinusing the crystalline form (I-HS).

FIG. 24 is a graph showing thermographic data for AM(HS)1. The top lineof the graph is a plot of the thermogravimetric analysis (TGA) for thecompound, while the bottom line is a plot of the differential scanningcalorimetry (DSC).

FIG. 25 is a graph showing thermographic data for AM(HS)2. The top lineof the graph is a plot of the thermogravimetric analysis (TGA) for thecompound, while the bottom line is a plot of the differential scanningcalorimetry (DSC).

FIG. 26 is a graph showing thermographic data for crystalline form(I-HS). The top line of the graph is a plot of the thermogravimetricanalysis (TGA) for the compound, while the bottom line is a plot of thedifferential scanning calorimetry (DSC).

FIG. 27 illustrates an overlay of the X-ray powder diffraction (XRPD)patterns of AM(HS)1, AM(HS)2, and crystalline form (I-HS). AM(HS)1 andAM(HS)2 are the broad lines in the lower part of the figure, whilecrystalline form (I-HS) exhibits sharp peaks.

FIG. 28 illustrates an X-ray powder diffraction (XRPD) pattern ofAM(HS)1 and AM(HS)2.

FIG. 29 illustrates an X-ray powder diffraction (XRPD) pattern ofcrystalline form (I-HS).

FIG. 30 is an image of a sample of AM(HS)1 under polarized lightmicroscopy at a magnification of 20×.

FIG. 31 is an image of a sample of AM(HS)2 under polarized lightmicroscopy at a magnification of 20×.

FIG. 32 is an image of a sample of crystalline form (I-HS) underpolarized light microscopy at a magnification of 20×.

FIG. 33 is a plot of the hygroscopicity of AM(HS)1 using dynamic vaporsorption (DVS).

FIG. 34 illustrates an X-ray powder diffraction (XRPD) pattern ofAM(HS)1 pre-DVS (top-line) and post-DVS (bottom line).

FIG. 35 is a plot of the hygroscopicity of AM(HS)2 using dynamic vaporsorption (DVS).

FIG. 36 illustrates an X-ray powder diffraction (XRPD) pattern ofAM(HS)2 pre-DVS (top-line) and post-DVS (bottom line).

FIG. 37 is a plot of the hygroscopicity of crystalline form (I-HS) usingdynamic vapor sorption (DVS).

FIG. 38 illustrates an X-ray powder diffraction (XRPD) pattern ofcrystalline form (I-HS) pre-DVS (top-line) and post-DVS (bottom line).

FIG. 39 is a plot of tensile strength versus compression pressure forvarious 200 mg direct compression blend compacts incorporatingcrystalline form (I-HS) or AM(HS)2. In the plot, (1) is a 2:1MCC:lactose blend with AM(HS)2; (2) is a 2:1 MCC:lactose blend withcrystalline form (I-HS); (3) is a 1:1 MCC:starch blend with AM(HS)2; (4)is a 1:1 MCC:starch blend with crystalline form (I-HS).

FIG. 40 is an overlay of DSC thermographs of AM(HS)1 at T0 (bottom line)and after 5 weeks at 40° C./75% RH (top line).

FIG. 41 is an overlay of DSC thermographs of crystalline form (I-HS) atT0 (bottom line) and after 5 weeks at 40° C./75% RH (top line).

FIG. 42 illustrates an overlay of the X-ray powder diffraction (XRPD)patterns of AM(HS)1 at T0 (broad line) and after 5 weeks at 40° C./75%RH (sharp peaks).

FIG. 43 illustrates an overlay of the X-ray powder diffraction (XRPD)patterns of crystalline form (I-HS) at T0 (bottom) and after 5 weeks at40° C./75% RH (top).

FIG. 44 illustrates an overlay of the X-ray powder diffraction (XRPD)patterns of crystalline form (I-HS) (bottom) and AM(HS)1 (top) after 5weeks at 40° C./75% RH.

FIG. 45 is a graph showing the percentage of change in volume of axenograph (human) tumor derived from a lung adenocarcinoma CUTO-3F cellline (CUTO-3.29) over time in mice that were treated with vehicle(triangles) or orally administered a daily dose of 60 mg/kg (circles) or200 mg/kg (squares) of crystalline form (I-HS) following implantation ofthe xenograft into the mice.

FIG. 46 is a graph showing the percentage of change in volume of axenograph (human) tumor derived from a colorectal cancer KM12 cell lineover time in mice that were treated with vehicle (triangles) or orallyadministered a daily dose of 60 mg/kg (circles) or 200 mg/kg (squares)of crystalline form (I-HS) following implantation of the xenograft intothe mice.

FIG. 47 is a graph showing the percentage of change in volume of axenograph (human) tumor derived from an acute myeloid leukemia MO-91cell line over time in mice that were treated with vehicle (triangles)or orally administered a daily dose of 60 mg/kg (circles) or 200 mg/kg(squares) of crystalline form (I-HS) following implantation of thexenograft into the mice.

The figures depict various embodiments of the present invention forpurposes of illustration only. One skilled in the art will readilyrecognize from the following discussion that alternative embodiments ofthe structures and methods illustrated herein may be employed withoutdeparting from the principles of the invention described herein.

DETAILED DESCRIPTION

The present disclosure relates to(S)—N-(5((R)-2-(2,5-difluorophenyl)-pyrrolidin-1-1)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide(Formula I) and to pharmaceutically acceptable salts thereof, forexample the hydrogen sulfate salt, and further to a novel crystallineform of the hydrogen sulfate salt, which exhibit Trk family proteintyrosine kinase inhibition, pharmaceutical compositions containing thesame, and processes of making the crystalline form.

Provided herein is a novel crystalline form of the compound of FormulaI:

In particular, the novel crystalline form comprises the hydrogen sulfatesalt of the compound of Formula I in a stable polymorph form,hereinafter referred to as crystalline form (I-HS), which may becharacterized, for example, by its X-ray diffraction pattern.

As illustrated in FIG. 1, in some embodiments, the crystalline form(I-HS) can be characterized by its X-ray powder diffraction pattern(XRPD). The XRPD was carried out on a D5000 X-ray diffractometer with aCuKα1, 0.1540562 nm long, fine focus sealed tube source from Siemens byscanning samples between 3 and 40° 2-theta at a step size of 0.0200°2-theta and a time per step of 1 second. The effective scan speed was0.0200°/s with an instrument voltage 40 kV and a current setting of 40mA. Samples were analyzed using a divergence slit having a size of 2 mmin reflection mode under the following experimental conditions.

In some embodiments, crystalline form (I-HS) has an XRPD pattern with atleast the 20 characteristic peaks (2θ degrees±0.3), as listed in Table1.

TABLE 1 XRPD peaks of crystalline form (I-HS) Relative Position [°2-θ]FWHM [°2-θ] d-spacing [Å] Intensity [%] 10.63 0.12 8.32 27.44 15.25 0.145.81 12.24 16.39 0.13 5.40 13.92 18.37 0.13 4.82 43.65 19.08 0.14 4.6519.60 19.79 0.11 4.48 9.83 20.15 0.25 4.40 25.09 20.61 0.13 4.31 100.0021.47 0.21 4.14 24.71 22.01 0.12 4.03 14.45 23.04 0.15 3.86 33.01 23.970.12 3.71 38.52 24.35 0.21 3.65 10.05 25.58 0.13 3.48 8.11 26.48 0.173.36 9.76 27.50 0.14 3.24 7.70 28.17 0.17 3.16 11.60 28.58 0.19 3.1210.85 30.77 0.29 2.90 8.48 38.47 0.21 2.34 10.97

In some embodiments, the crystalline form (I-HS) has an XRPD patternwith at least the 8 characteristic peaks (2θ degrees±0.3), whichcomprises peaks having a relative intensity greater than or equal toabout 15%, as listed in Table 2.

TABLE 2 XRPD peaks of crystalline form (I-HS) Relative Position [°2-θ]FWHM [°2-θ] d-spacing [Å] Intensity [%] 10.63 0.12 8.32 27.44 18.37 0.134.82 43.65 19.08 0.14 4.65 19.60 20.15 0.25 4.40 25.09 20.61 0.13 4.31100.00 21.47 0.21 4.14 24.71 23.04 0.15 3.86 33.01 23.97 0.12 3.71 38.52

In some embodiments, the crystalline form (I-HS) has an XRPD patternwith at least the 5 characteristic peaks (2θ degrees±0.3), whichcomprises peaks having a relative intensity greater than or equal toabout 25%, as listed in Table 3.

TABLE 3 XRPD peaks of crystalline form (I-HS) Relative Position [°2-θ]FWHM [°2-θ] d-spacing [Å] Intensity [%] 10.63 0.12 8.32 27.44 18.37 0.134.82 43.65 20.61 0.13 4.31 100.00 23.04 0.15 3.86 33.01 23.97 0.12 3.7138.52

In some embodiments, the crystalline form (I-HS) has an XRPD patternwith at least the 4 characteristic peaks (2θ degrees±0.3), whichcomprises peaks having a relative intensity greater than or equal toabout 30%, as listed in Table 4.

TABLE 4 XRPD peaks of crystalline form (I-HS) Relative Position [°2-θ]FWHM [°2-θ] d-spacing [Å] Intensity [%] 18.37 0.13 4.82 43.65 20.61 0.134.31 100.00 23.04 0.15 3.86 33.01 23.97 0.12 3.71 38.52

In certain embodiments, crystalline form (I-HS) has an XRPD pattern thatis substantially the same XRPD pattern as shown in FIG. 1.

In some embodiments, crystalline form (I-HS) is characterized by havingXRPD diffraction peaks (2θ degrees) at about 18.4, 20.6, 23.0, and 24.0.In some embodiments, crystalline form (I-HS) is characterized by havingXRPD diffraction peaks (2θ degrees) at about 10.6, 18.4, 20.6, 23.0, and24.0. In some embodiments, crystalline form (I-HS) is characterized byhaving XRPD diffraction peaks (2θ degrees) at about 10.6, 18.4, 19.1,20.2, 20.6, 21.5, 23.0, and 24.0. In some embodiments, crystalline form(I-HS) is characterized by having XRPD diffraction peaks (2θ degrees) atabout 10.6, 15.3, 16.4, 18.4, 19.1, 19.8, 20.2, 20.6, 21.5, 22.0, 23.0,24.0, 24.4, 25.6, 26.5, 27.5, 28.2, 28.6, 30.8, and 38.5.

In certain embodiments, crystalline form (I-HS) has an XRPD pattern thatis substantially the same XRPD pattern as shown in FIG. 29.

In some embodiments, crystalline form (I-HS) has an XRPD pattern with atleast the 20 characteristic peaks (2θ degrees±0.3), as listed in Table1.

TABLE 5 XRPD peaks of crystalline form (I-HS) Position (°2θ) RelativeIntensity (%) 10.76 29.85 15.38 13.22 16.52 16.46 18.50 48.07 19.2222.92 19.92 16.05 20.26 30.80 20.74 100.00 21.56 23.78 22.16 15.51 23.1632.52 24.10 33.89 24.50 12.14 25.72 8.89 26.50 10.88 27.62 8.61 28.3211.44 28.74 10.73 30.92 8.23 38.60 8.88

In some embodiments, the crystalline form (I-HS) has an XRPD patternwith at least the 8 characteristic peaks (2θ degrees±0.3), whichcomprises peaks having a relative intensity greater than or equal toabout 15%, as listed in Table 6.

TABLE 6 XRPD peaks of crystalline form (I-HS) Position (°2θ) RelativeIntensity (%) 10.76 29.85 18.50 48.07 19.22 22.92 20.26 30.80 20.74100.00 21.56 23.78 23.16 32.52 24.10 33.89

In some embodiments, the crystalline form (I-HS) has an XRPD patternwith at least the 5 characteristic peaks (2θ degrees±0.3), whichcomprises peaks having a relative intensity greater than or equal toabout 25%, as listed in Table 7.

TABLE 7 XRPD peaks of crystalline form (I-HS) Position (°2θ) RelativeIntensity (%) 10.76 29.85 18.50 48.07 20.74 100.00 23.16 32.52 24.1033.89

In some embodiments, the crystalline form (I-HS) has an XRPD patternwith at least the 4 characteristic peaks (2θ degrees±0.3), whichcomprises peaks having a relative intensity greater than or equal toabout 30%, as listed in Table 8.

TABLE 8 XRPD peaks of crystalline form (I-HS) Position (°2θ) RelativeIntensity (%) 18.50 48.07 20.74 100.00 23.16 32.52 24.10 33.89

In some embodiments, crystalline form (I-HS) is characterized by havingXRPD diffraction peaks (2θ degrees) at about 18.5, 20.7, 23.2, and 24.1.In some embodiments, crystalline form (I-HS) is characterized by havingXRPD diffraction peaks (2θ degrees) at about 10.8, 18.5, 20.7, 23.2, and24.1. In some embodiments, crystalline form (I-HS) is characterized byhaving XRPD diffraction peaks (2θ degrees) at about 10.8, 18.5, 19.2,20.3, 20.7, 21.6, 23.2, and 24.1. In some embodiments, crystalline form(I-HS) is characterized by having XRPD diffraction peaks (2θ degrees) atabout 10.8, 15.4, 16.5, 18.5, 19.2, 19.9, 20.3, 20.7, 21.6, 22.2, 23.2,24.1, 24.5, 25.7, 26.5, 27.6, 28.3, 28.7, 30.9, and 38.6.

In some embodiments, given the XRPD patterns provided in FIGS. 1 and 29,crystalline form (I-HS) is characterized by having XRPD peaks (2θdegrees) as shown in Table 9.

TABLE 9 XRPD peaks of crystalline form (I-HS) FIG. 1 FIG. 29 DifferenceAverage 10.76 10.63 0.13 10.70 15.38 15.25 0.13 15.32 16.52 16.39 0.1316.46 18.50 18.37 0.13 18.44 19.22 19.08 0.14 19.15 19.92 19.79 0.1319.86 20.26 20.15 0.11 20.21 20.74 20.61 0.13 20.68 21.56 21.47 0.0921.52 22.16 22.01 0.15 22.09 23.16 23.04 0.12 23.10 24.10 23.97 0.1324.04 24.50 24.35 0.15 24.43 25.72 25.58 0.14 25.65 26.50 26.48 0.0226.49 27.62 27.50 0.12 27.56 28.32 28.17 0.15 28.25 28.74 28.58 0.1628.66 30.92 30.77 0.15 30.85 38.60 38.47 0.13 38.54

In some embodiments, crystalline form (I-HS) is characterized by havingXRPD diffraction peaks (2θ degrees) at 18.4±0.2, 20.7±0.2, 23.1±0.2, and24.0±0.2. In some embodiments, crystalline form (I-HS) is characterizedby having XRPD diffraction peaks (20 degrees) at 10.7±0.2, 18.4±0.2,20.7±0.2, 23.1±0.2, and 24.0±0.2. In some embodiments, crystalline form(I-HS) is characterized by having XRPD diffraction peaks (2θ degrees) at10.7±0.2, 18.4±0.2, 19.2±0.2, 20.2±0.2, 20.7±0.2, 21.5±0.2, 23.1±0.2,and 24.0±0.2. In some embodiments, crystalline form (I-HS) ischaracterized by having XRPD diffraction peaks (2θ degrees) at 10.7±0.2,15.3±0.2, 16.5±0.2, 18.4±0.2, 19.2±0.2, 19.9±0.2, 20.2±0.2, 20.7±0.2,21.5±0.2, 22.1±0.2, 23.1±0.2, 24.0±0.2. 24.4±0.2, 25.6±0.2, 26.5±0.2,27.6±0.2, 28.2±0.2, 28.7±0.2, 30.8±0.2, and 38.5±0.2.

It will be understood that the 2-theta values of the X-ray powderdiffraction patterns for crystalline form (I-HS) may vary slightly fromone instrument to another and also depending on variations in samplepreparation and batch to batch variation, and so the values quoted arenot to be construed as absolute. It will also be understood that therelative intensities of peaks may vary depending on orientation effectsso that the intensities shown in the XRPD trace included herein areillustrative and not intended to be used for absolute comparison.Accordingly, it is to be understood that the phrase “substantially thesame XRPD pattern as shown in FIG. 1 or FIG. 29” means that forcomparison purposes, at least 90% of the peaks shown in FIG. 1 or FIG.29 are present. It is to be understood that the relative peak positionsmay vary ±0.3 degrees from the peak positions shown in FIG. 1 or FIG.29. It is to be further understood that for comparison purposes somevariability in peak intensities from those shown in FIG. 1 and FIG. 29is allowed.

FIG. 2 illustrates a simultaneous thermogravimetric/differential thermalanalyzer (TG/DTA) profile of crystalline form (I-HS), according to oneembodiment. For the analysis about 5 mg of crystalline form (I-HS) wasweighed into an open aluminum pan and loaded into a simultaneousthermogravimetric/differential thermal analyzer (TG/DTA) and held atroom temperature. The sample was then heated at a rate of 10°Celsius/min from 25° Celsius to 300° Celsius during which time thechange in sample weight was recorded along with any differential thermalevents. Nitrogen was used as the purge gas at a flow rate of 100cm³/min. The TG/DAT profile of crystalline form (I-HS) shows an initialweight loss of 0.8% between 27.4° Celsius to 182.4° Celsius, which isfollowed by 4.9% weight loss in the TG curve between 182.4° Celsius to225.0° Celsius, also seen as an endotherm in the DTA curve. These weightlosses could be decomposition of the material.

FIG. 3 illustrates a differential scanning calorimetry (DSC) profile ofcrystalline form (I-HS), according to one embodiment. DSC analysis ofthe sample was performed using a Seiko DSC6200 differential scanningcalorimeter (equipped with a cooler). About 5 mg of crystalline form(I-HS) was weighed into an aluminum DSC pan and sealed non-hermeticallywith a pierced aluminum lid. The sample pan was then loaded into a SeikoDSC6200 (equipped with a cooler), cooled, and held at 25° Celsius. Oncea stable heat-flow response was obtained, the sample and reference wereheated to 270° Celsius at a scan rate of 10° Celsius/min whilemonitoring the resulting heat flow response. In some embodiments,crystalline form (I-HS) has a DSC thermogram substantially as shown inFIG. 3. As used herein, “substantially as shown in FIG. 3” means thatthe temperatures of the endothermic event shown in FIG. 3 can vary byabout ±5° C.

As shown in FIG. 3, the DSC thermogram of the crystalline form (I-HS)indicates a small endothermic change in the baseline between 122.9°Celsius to 152.8° Celsius, followed by a sharp endotherm thatcorresponds to the melting of the crystalline form (I-HS) at an onsettemperature of melting of 190.8° Celsius, a peak temperature of meltingof 197.9° Celsius and a heat of melting of 2.415 mW. The transitionfollowing the melting endotherm may be caused by the decomposition ofthe melted crystalline form (I-HS).

FIGS. 4A and 4B illustrate polarized light microscopy (PLM) images ofcrystalline form (I-HS) under (A) unpolarized and (B) unpolarized light,according to some embodiments. The presence of crystallinity(birefringence) was determined using an Olympus BX50 polarizingmicroscope, equipped with a Motic camera and image capture software(Motic Images Plus 2.0). All images were recorded using the 20×objective. The crystalline form (I-HS) exhibits birefringence whenexamined under polarized light without exhibiting a definite morphologyor agglomerates.

FIG. 5 illustrates a dynamic vapor sorption (DVS) isotherm profile ofcrystalline form (I-HS), according to one embodiment. For the DVSmeasurement a sample of crystalline form (I-HS) was cycled throughchanging humidity conditions to determine its hygroscopicity. The samplewas analyzed using a Surface Measurement System DVS-1 Dynamic VaporSorption System. About 10 mg of crystalline form (I-HS) was placed intoa mesh vapor sorption balance pan and loaded into a dynamic vaporsorption balance as part of the Surface Measurement System. Data wascollected in 1 minute intervals. Nitrogen was used as the carrier gas.The sampled crystalline form (I-HS) was subjected to a ramping profilefrom 20-90% relative humidity (RH) at 10% increments, maintaining thesample at each step until a stable weight had been achieved (99.5% stepcompletion). After completion of the sorption cycle, the sample wasdried using the same procedure, but all the way down to 0% RH andfinally taken back to the starting point of 20% RH. The weight changeduring the sorption/desorption cycles were plotted, allowing for thehygroscopic nature of the sample to be determined.

As shown in FIG. 5, crystalline form (I-HS) appears to benon-hygroscopic. A small increase in mass of about 1.7% was observedbetween 0% and 90% RH during the sorption cycle. In addition, a verysmall hysteresis was observed between sorption and desorption cycles.The XRPD pattern of crystalline form (I-HS) post DVS analysis (notshown) being similar to its pre-DVS XRPD pattern shown in FIG. 1 or FIG.29 indicates that no change in the crystalline form (I-HS) occurredduring DVS.

FIG. 6 illustrates an infrared (IR) spectroscopy profile of crystallineform (I-HS) for the compound of Formula I, according to one embodiment.IR spectroscopy was carried out on a Bruker ALPHA P spectrometer.Sufficient material of crystalline form (I-HS) was placed onto thecenter of the plate of the spectrometer with a transmittance spectrumbeing obtained using a resolution of 4 cm⁻¹, a background scan time of16 scans, a sample scan time of 16 scans, and collecting data from 4000cm⁻¹ to 400 cm⁻¹. The observed IR spectrum of crystalline form (I-HS) isshown in FIG. 6.

The crystalline form (I-HS) has a number of properties that make itsurprisingly superior to the amorphous form of(S)—N-(5((R)-2-(2,5-difluorophenyl)-pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehydrogen sulfate (AM(HS)). For example, the crystalline form (I-HS) hasproperties which contribute to its manufacturability and production of acommercial product. As shown in Example 8, the crystalline form (I-HS)has better flow properties as compared to the amorphous API (AM(HS)) asevidenced by the Carr's and Hausner Index. For example, the crystallineform (I-HS) exhibits a Can Index value of greater than 20%. In someembodiments, the crystalline form (I-HS) exhibits a Hausner ratio ofless than 1.35 (e.g., a value of between about 1.26 to about 1.34). Thedifferences in flow properties can make the development of a solid oraldosage form more difficult for the amorphous API vs. the crystallineAPI.

The crystalline form (I-HS) also evidenced better stability in anaccelerated stability study conducted in an LDPE bag at 40° C./75% RHfor five weeks. While neither the AM(HS) or crystalline form (I-HS)exhibited a significant changes in chemical impurity levels over thecourse of the study, the study did reveal that the crystalline form(I-HS) has stable physicochemical properties. The amorphous API, on theother hand, converted into a crystalline form substantially similar tothe crystalline form (I-HS) by XRPD, DSC, TGA, KF and polarized lightmicroscopy. Additionally, the amorphous API changed to an agglomeratedpowder with reduced flow properties over the course of the stabilitytesting. Such changes in the physical properties of the compound,including a change from an amorphous power to a crystalline materialand/or an agglomerated powder with reduced flow, on storage would makeit nearly impossible to manufacture a solid oral dosage form for patientuse based on the amorphous compound. The properties observed for thecrystalline form (I-HS), however, are consistent with that desired for acommercial product, including having both a stable physical and chemicalstructure.

The crystalline form (I-HS), as noted previously, is non-hygroscopic. Asused herein, “non-hygroscopic” refers to a compound exhibiting less thana 2% weight gain at 25° C. and 80% RH after 24 to 48 hours (see, e.g.,Example 10). The AM(HS) compound, however, was found to deliquesce uponexposure to humidity. Given this tendency, use of the AM(HS) compoundwould require significant handling precautions during storage andmanufacture to prevent this change in form from occurring whereas thecrystalline form (I-HS) requires no such precautions during manufactureof the API. This stability to humidity would also be expected to carryover to any solid oral dosage product prepared using the crystallineform (I-HS).

Finally, the crystalline form (I-HS) provides a significantly improvedimpurity profile versus the amorphous API. The ability to control animpurity profile is important for patient safety, developing arepeatable manufacturing process, and meeting requirements by Regulatoryagencies prior to use in humans.

The compounds provided herein, including(S)—N-(5((R)-2-(2,5-difluorophenyl)-pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide(Formula I) and pharmaceutically acceptable salts thereof, for examplethe hydrogen sulfate salt, and further a novel crystalline form of thehydrogen sulfate salt (crystalline form (I-HS)), exhibit Trk familyprotein tyrosine kinase inhibition, and the compound, hydrogen sulfatesalt, and crystalline form thereof can be used in the treatment of pain,inflammation, cancer, and certain infectious diseases.

Some embodiments include the use of the crystalline form (I-HS) for thetreatment of disorders and diseases which can be treated by inhibitingTrkA, TrkB and/or TrkC kinases, such as a TrkA, TrkB and/or TrkCmediated condition, such as one or more conditions described herein,including a Trk-associated cancer. In some embodiments, the crystallineform (I-HS) may be also useful in the treatment of pain, includingchronic and acute pain. In some embodiments, the crystalline form (I-HS)may be useful in the treatment of multiple types of pain includinginflammatory pain, neuropathic pain, surgical pain, and pain associatedwith cancer, surgery and bone fracture. In addition, the crystallineform (I-HS) may be useful for treating inflammation, active and chronicneurodegenerative diseases and certain infectious diseases. The presentdisclosure is further directed to pharmaceutical compositions comprisingcrystalline form (I-HS). In some embodiments, the pharmaceuticalcomposition comprises crystalline form (I-HS) and a pharmaceuticallyacceptable diluent or carrier.

The ability of crystalline form (I-HS) to act as TrkA, TrkB and/or TrkCinhibitors may be demonstrated by the assays described in Examples A andB as disclosed in U.S. Pat. No. 8,513,263, issued on Aug. 20, 2013,which is incorporated herein by reference.

In some embodiments, provided herein is a method for treating a patientdiagnosed with a TRK-associated cancer, comprising administering to thepatient a therapeutically effective amount of crystalline form (I-HS) ora compound of Formula I or a salt thereof, such as a hydrogen sulfatesalt (e.g., see Example 14A of U.S. Pat. No. 8,513,263). Trk family ofneurotrophin receptors, TrkA, TrkB, and TrkC (encoded by NTRK1, NTRK2,and NTRK3 genes, respectively) and their neurotrophin ligands regulategrowth, differentiation and survival of neurons. Dysregulation in a NTRKgene, a Trk protein, or expression or activity, or level of the same,such as translocations involving the NTRK kinase domain, mutationsinvolving the TRK ligand-binding site, amplifications of a NTRK gene,Trk mRNA splice variants, and Trk autocrine/paracrine signaling aredescribed in a diverse number of tumor types and may contribute totumorigenesis. Recently NTRK1 fusions were described in a subset ofadenocarcinoma lung cancer patients². Translocations in NTRK1, NTRK2,and NTRK3 that lead to the production of constitutively-active TrkA,TrkB, and TrkC fusion proteins are oncogenic and prevalent in a widearray of tumor types, including lung adenocarcinoma, thyroid, head andneck cancer, glioblastoma, and others.

In some embodiments, the dysregulation in a NTRK gene, a Trk protein, orexpression or activity, or level of the same, includes overexpression ofwild-type TrkA, TrkB, or TrkC (e.g., leading to autocrine activation).In some embodiments, the dysregulation in a NTRK gene, a Trk protein, orexpression or activity, or level of the same, includes overexpression,activation, amplification or mutation in a chromosomal segmentcomprising the NTRK1, NTRK2, or NTKR3 gene or a portion thereof In someembodiments, the dysregulation of a NTRK gene, a Trk protein, orexpression or activity, or level of the same, includes one or morechromosome translocations or inversions resulting in NTRK1, NTRK2, orNTRK3 gene fusions, respectively. In some embodiments, the dysregulationof a NTRK gene, a Trk protein, or expression or activity, or level ofthe same, is a result of genetic translocations in which the expressedprotein is a fusion protein containing residues from a non-TrkA partnerprotein and TrkA, a non-TrkB partner protein and TrkB, or a non-TrkCpartner protein and TrkC proteins, and include a minimum of a functionalTrkA, TrkB, or TrkC kinase domain, respectively.

In some embodiments, a TrkA fusion protein is one of the TrkA fusionproteins shown in Table 10, where:

TABLE 10 Exemplary TrkA Fusion Proteins and Cancers Non-limitingExemplary Trk- and Synonyms of Associated Fusion Protein Non-TrkA FusionPartner Cancer(s) TP53-TrkA^(1,11) Tumor Protein P53 Spitzoid Melanoma,Spitz tumors LMNA-TrkA^(1, 12) Lamin A/C Spitzoid Melanoma, Spitztumors, Undifferentiated Sarcoma, Adult Soft Tissue Sarcoma (e.g., SoftTissue Sarcoma Metastatic to Lung), Soft Tissue Fibrosarcoma CD74-TrkA²MHC class II invariant chain Non-Small Cell Lung Cancer (NSCLC) Lungadenocarcimona TFG-TrkA (TRK- TRK-Fused Gene Papillary Thyroid CarcinomaT3)³ (PTC), Soft Tissue Solitary Fibrous Tumor TPM3-TrkA³ Tropomyosin 3Lung Cancer, Papillary Thyroid Carcinoma (PTC), Acute Myeloid Leukemia(AML), Sarcoma, Pediatric Gliomas, Colorectal Cancer (CRC), Soft TissueSchwannoma NFASC-TrkA⁴ Neurofascin Gliobastoma multiforme (GBM);Glioblastoma BCAN-TrkA⁴ Brevican Glioblastoma multiforme (GBM)MPRIP-TrkA⁵ Myosin Phosphatase Rho Non-small cell lung cancerInteracting Protein or Rho (NSCLC), Lung adenocarcinoma InteractingProtein 3 TPR-TrkA (TRK- Translocated Promoter Region, Papillary ThyroidCarcinoma T1 or TRK-T2)³ Nuclear Basket Protein (PTC), Colorectal Cancer(CRC)^(A), Non-small cell lung cancer (NSCLC) RFWD2-TrkA⁶ Ring Fingerand WD Repeat Large Cell Neuroendrocine Cancer Domain 2 (LCNEC); NSCLCIRF2BP2-TrkA⁷ Interferon Regulatory Factor 2 Thyroid Cancer; ThyroidGland Binding Protein 2 Carcinoma SQSTM1-TrkA⁷ Sequestosome 1 ThyroidCancer (e.g., Papillary Thyroid Cancer), Thyroid Gland Carcinoma, SoftTissue Fibrosarcoma SSBP2-TrkA⁷ Single-Stranded DNA Binding ThyroidCancer (e.g., Papillary Protein 2 Thyroid Cancer); Thyroid GlandCarcinoma RABGAP1L- RAB GTPase Activating Intrahepatic CholangicarcinomaTrkA⁸ Protein 1-Like (ICC) C18ORF8-TrkA⁹ Chromosome 18 Open ReadingNon-Small Cell Lung Cancer Frame 8 (NSCLC) RNF213-TrkA⁹ Ring FingerProtein 213 Non-Small Cell Lung Cancer (NSCLC) TBC1D22A- TBC1 DomainFamily, Member Non-Small Cell Lung Cancer TrkA⁹ 22A (NSCLC) C20ORF112-Chromosome 20 Open Reading Non-Small Cell Lung Cancer TrkA⁹ Frame 112(NSCLC) DNER-TrkA⁹ Delta/Notch-Like EGF Repeat Non-Small Cell LungCancer Containing (NSCLC) ARHGEF2- Rho Guanine Nucleotide GlioblastomaTrkA¹³ Exchange Factor 2 CHTOP-TrkA¹³ Chromatin Target of PRMT1Glioblastoma PPL-TrkA¹³ Periplakin Thyroid Carcinoma PLEKHA6-TrkAPleckstrin Homology Domain- Containing Family A Member 6 PEAR1-TrkAPlatelet Endothelial Aggregation Receptor 1 MRPL24-TrkA 39S RibosomalProtein L24, Mitochondrial MDM4-TrkA Human Homolg of Mouse Double Minute4 LRRC71-TrkA Leucine Rich Repeat Containing 71 GRIPAP1-TrkA GRIP1Associated Protein 1 EPS15-TrkA Epidermal Growth Factor ReceptorSubstrate 15 DYNC2H1- Dynein, Cytoplasmic 2, Heavy Sarcoma TrkA^(B)Chain 1 CEL-TrkA Carboxyl Ester Lipase Pancreatic adenocarcinomasample^(D) EPHB2-TrkA^(B) EPH Receptor B2 Lower Grade GliomaTGF-TrkA^(C) Transforming Growth Factor Papillary Thyroid Cancer^(A)Créancier et al., Cancer Lett. 365(1): 107-111, 2015. ^(B)U.S.Patent Application Publication No. 2015/0315657. ^(C)U.S. PatentApplication Publication No. 2015/0283132. ^(D)Egren et al., Cancer Res.75(15 Supplement): 4793, 2015.

In some embodiments, the dysregulation of a NTRK gene, a Trk protein, orexpression or activity, or level of the same, includes one or moredeletions, insertions, or point mutation(s) in a TrkA protein. In someembodiments, the dysregulation of a NTRK gene, a Trk protein, orexpression or activity, or level of the same, includes a deletion of oneor more residues from the TrkA protein, resulting in constitutiveactivity of the TrkA kinase domain. In some embodiments, the deletionincludes a deletion of amino acids 303-377 in TrkA isoform 2.

In some embodiments, the dysregulation of a NTRK gene, a Trk protein, orexpression or activity, or level of the same, includes at least onepoint mutation in a NTRK1 gene that results in the production of a TrkAprotein that has one or more amino acid substitutions as compared to thewildtype TrkA protein (see, for example, the point mutations listed inTable 11.

TABLE 11 Activating TrkA Point Mutations Point Mutation Rationale R33W¹A336E Near NGF Binding Site A337T Near NGF Binding Site R324Q or R324WNear NGF Binding Site V420M Close to Membrane R444Q or R444W Close toMembrane G517R or G517V P-Loop K538A Activating R649W or R649L Argininemay stabilize auto- inhibited conformation. R682S Activation Loop V683GActivation Loop R702C Exposed, may form face-to-face disulfide linkeddimer C1879T² ¹Zhang et al., Blood 124(21): 1682, 2014. Mutation foundin T-cell prolymphocytic leukemia. ²Park et al., Proc. Natl. Acad. Sci.U.S.A. 112(40): 12492-12497, 2015. Mutation found in colorectal cancer.

In some embodiments, the dysregulation of a NTRK gene, a Trk protein, orexpression or activity, or level of the same, includes a splicevariation in a TrkA mRNA which results in an expressed protein that isan alternatively spliced variant of TrkA having at least one residuedeleted (as compared to a wild-type TrkA protein) resulting inconstitutive activity of the TrkA kinase domain. In some embodiments, analternatively spliced form of TrkA with constitutive activity hasdeletions of exons 8, 9, and 11 resulting in an expressed proteinmissing residues 192-284 and 393-398 relative to TrkA Isoform 2, has adeletion of exon 10 in TrkA, or has a deletion in a NTRK1 gene thatencodes a TrkA protein with a 75 amino acid deletion in thetransmembrane domain (Reuther et al., Mol. Cell Biol. 20:8655-8666,2000).

Cancers identified as having dysregulation of a NTRK gene, a Trkprotein, or expression or activity, or level of the same, (seereferences cited herein and also the www.cancer.gov and www.nccn.orgwebsites) include:

(A) Cancers wherein the dysregulation of a NTRK gene, a Trk protein, orexpression or activity, or level of the same, includes one or morechromosome translocations or inversions resulting in TrkA fusionproteins, e.g., including:

Cancer Standard of Care Non-Small Cell radiotherapy (e.g., radioiodidetherapy, external-beam Lung Cancer² radiation, or radium 223 therapy),chemotherapeutics as single agents (e.g., afatinib dimaleate,bevacizumab, carboplatin, cetuximab, cisplatin, crizotinib, erlotinib,gefitinib, gemcitabine, methotrexate, paclitaxel, or pemetrexed) orcombinations (e.g., carboplatin- paclitaxel, gemcitabine-paclitaxel, orchemoradiation) Papillary Radiotherapies (e.g., radioiodide therapy orThyroid external-beam radiation) and chemotherapeutics Carcinoma¹⁴(e.g., sorafenib, sunitinib, or pazopanib) GlioblastomaChemotherapeutics (e.g., bevacizumab, everolimus, Multiforme¹⁵lomustine, or temozolomide) Colorectal Chemotherapeutics as singleagents (e.g., aflibercept, Carcinoma¹⁶ bevacizumab, capecitabine,cetuximab, fluorouracil, irinotecan, leucovorin, oxaliplatin,panitumumab, or regorafenib) or combinations (e.g., folfox, folfiri,capox, folfiri-bevacizumab, folfiri-cetuximab, or xelox) Melanoma¹²Chemotherapeutics (e.g., aldesleukin, dabrafenib, dacarbazine,interferon alfa-2b, ipilimumab, peginterferon alfa-2b, trametinib, orvemurafenib)

(B) Cancers wherein the dysregulation of a NTRK gene, a Trk protein, orexpression or activity, or level of the same, includes one or moredeletions, insertions, or mutations in the TrkA protein, e.g.,including:

Cancer Standard of care Acute Myeloid Chemotherapeutics as single agents(e.g., leukemia^(17, 18) arsenic trioxide, cyclophosphamide, cytarabine,daunorubicin, doxorubicin, or vincristine) or combinations (e.g., ADE)Large Cell Radiotherapy (e.g., radioiodide therapy, external-beamNeuroendocrine radiation, or radium 223 therapy) and/or chemother-Carcinoma¹⁹ apeutics (e.g., cisplatin, carboplatin, or etoposide)Neuroblastoma²⁰ Chemotherapeutics (e.g., cyclophosphamide, doxorubicin,or vincristine)

(C) Cancers wherein the dysregulation of a NTRK gene, a Trk protein, orexpression or activity, or level of the same, includes overexpression ofwildtype TrkA (autocrine activation), e.g., including:

Cancer Standard of care Prostate Radiotherapy (e.g., radium 223 therapy)or Carcinoma^(21, 22) chemotherapeutics (e.g. abiraterone, cabazitaxel,degarelix, denosumab, docetaxel, enzalutamide, leuprolide, prednisone,or sipuleucel-T) Neuroblastoma²³ Chemotherapeutics (e.g.,cyclophosphamide, doxorubicin, or vincristine) PancreaticChemotherapeutics as single agents (e.g., erlotinib, Carcinoma²⁴fluorouracil, gemcitabine, or mitomycin C) or combinations (e.g.,gemcitabine-oxaliplatin) Melanoma²⁵ Chemotherapeutics (e.g.,aldesleukin, dabrafenib, dacarbazine, interferon alfa-2b, ipilimumab,peginterferon alfa-2b, trametinib, or vemurafenib) Head and NeckRadiotherapy and/or chemotherapeutics (e.g., Squamous Cell bleomycin,cetuximab, cisplatin, docetaxel, Carcinoma²⁶ fluorouracil, ormethotrexate) Gastric Chemotherapeutics (e.g., docetaxel, Carcinoma²⁷doxorubucin, fluorouracil, mitomycin C, or trastuzumab)

In some embodiments, the dysregulation of a NTRK gene, a Trk protein, orexpression or activity, or level of the same, includes a translocationthat results in the expression of a TrkB fusion protein, e.g., one ofthe TrkB fusion proteins shown in Table 12.

TABLE 12 Exemplary TrkB Fusion Proteins and Cancers Non-limitingExemplary Trk- and Synonyms of Fusion Protein Non-TrkB Fusion PartnerAssociated Cancer(s) NACC2-TrkB¹⁰ NACC Family Member 2, PilocyticAstrocytoma BEN and BTB (POZ) Domain Containing QKI-TrkB¹⁰ QKI, KHDomain Pilocytic Astrocytoma Containing, RNA Binding AFAP1-TrkB⁷ ActinFilament Associated Lower-grade Glioma Protein 1 PAN3-TrkB⁷ PAN3 Poly(A)Specific Head and Neck Ribonuclease Subunit Squamous Cell CarcinomaSQSTM1-TrkB⁷ Sequestosome 1 Lower-Grade Glioma TRIM24-TrkB⁷ TripartiteMotif Lung adenocarcinoma Containing 24 VCL-TrkB¹¹ Vinculin Pediatricgliomas AGBL4-TrkB¹¹ ATP/GTP Binding Pediatric gliomas Protein-Like 4DAB2IP-TrkB Disabled Homolog 2- Interacting Protein

In some embodiments, the dysregulation of a NTRK gene, a Trk protein, orexpression or activity, or level of the same, includes a translocationwhich results in the expression of a TrkC fusion protein, e.g., one ofthe TrkC fusion proteins shown in Table 13.

TABLE 13 Exemplary TrkC Fusion Proteins and Cancers Non-limitingExemplary Trk- Non-TrkB Fusion and Synonyms of Associated Fusion ProteinPartner Cancer(s) ETV6-TrkC¹¹ ETS Variant 6 Salivary Gland Cancer,Secretory Breast Carcinoma, Acute Myeloid Leukemia, Fibrosarcoma,Nephroma, Melanoma, Colorectal Cancer (CRC), Breast Cancer, PediatricGliomas, Thyroid Cancer (e.g., Papillary Thyroid Cancer), InfantileFibrosarcoma, Soft Tissue Hemangioma, Gastrointestinal Stromal Tumor(GIST) (e.g., c-kit-negative GIST), Mammary Carcinoma (e.g., MammaryAnalogue Secretory Carcinoma) BTBD1-TrkC¹¹ BTB (POZ) Domain PediatricGliomas Containing 1 LYN-TrkC⁷ V-Yes-1 Yamaguchi Head and Neck SquamousSarcoma Viral Related Cell Carcinoma Oncogene Homolog (also known asLck/Yes-Related Novel Protein Tyrosine Kinase) RBPMS-TrkC⁷ RNA BindingProtein Thyroid Cancer with Multiple (e.g., Papillary Splicing ThyroidCancer) EML4-TrkC^(B) Echinoderm Fibrosarcoma Microtubule- AssociatedProtein- Like 4 HOMER2-TrkC Homer Protein Soft Tissue Sarcoma Homolog 2TFG-TrkC TRK-Fused Gene Soft Tissue Solitary Fibrous Tumor FAT1-TrkCCervical Squamous Cell Carcinoma^(C) TEL-TrkC Congenital Fibrosarcoma,Acute Myelogenous Leukemia ^(B)Tannenbaum et al., Cold Spring Harb. Mol.Case Stud. 1: a000471, 2015. ^(C)U.S. Patent Application Publication No.2015/0315657.

In some embodiments, provided herein is a method for treating a patientdiagnosed with a Trk-associated cancer, comprising administering to thepatient a therapeutically effective amount of crystalline form (I-HS) ora compound of Formula I or a salt thereof, such as a hydrogen sulfatesalt (e.g., see Example 14A of U.S. Pat. No. 8,513,263). For example,the Trk-associated cancer can be selected from the group of: non-smallcell lung cancer, papillary thyroid carcinoma, glioblastoma multiforme,acute myeloid leukemia, colorectal carcinoma, large cell neuroendocrinecarcinoma, prostate cancer, neuroblastoma, pancreatic carcinoma,melanoma, head and neck squamous cell carcinoma, gastric carcinoma,Spitz cancer, papillary thyroid carcinoma, colon cancer, acute myeloidleukemia, sarcoma, pediatric glioma, intrahepatic cholangicarcinoma,pilocytic astrocytoma, lower grade glioma, lung adenocarcinoma, salivarygland cancer, secretory breast cancer, fibrosarcoma, nephroma, andbreast cancer.

In some embodiments, a Trk-associated cancer is selected from the groupof: non-limiting examples of TRK-associated cancers include: Spitzoidmelanoma, Spitz tumors (e.g., metastatic Spitz tumors), non-small celllung cancer (NSCLC), thyroid carcinoma (e.g., papillary thyroidcarcinoma (PTC)), acute myeloid leukemia (AML), sarcoma (e.g.,undifferentiated sarcoma or adult soft tissue sarcoma), pediatricgliomas, colorectal cancer (CRC), gliobastoma multiforme (GBM), largecell neuroendocrine cancer (LCNEC), thyroid cancer, intrahepaticcholangicarcinoma (ICC), pilocytic astrocytoma, lower-grade glioma, headand neck squamous cell carcinoma, adenocarcinoma (e.g., lungadenocarcinoma), salivary gland cancer, secretory breast carcinoma,breast cancer, acute myeloid leukemia, fibrosarcoma, nephroma, melanoma,bronchogenic carcinoma, B-cell cancer, Bronchus cancer, cancer of theoral cavity or pharynx, cancer of hematological tissues, cervicalcancer, gastric cancer, kidney cancer, liver cancer, multiple myeloma,ovarian cancer, pancreatic cancer, salivary gland cancer, small bowel orappendix cancer, testicular cancer, urinary bladder cancer, uterine orendrometrial cancer, inflammatory myofibroblastic tumors,gastrointestinal stromal tumor, non-Hodgkin's lymphoma, neuroblastoma,small cell lung cancer, squamous cell carcinoma, esophageal-gastriccancer, skin cancer, neoplasm (e.g., a melanocystic neoplasm), Spitznevi, astrocytoma, medulloblastoma, glioma, large cell neuroendocrinetumors, bone cancer, and rectum carcinoma.

In some embodiments, the compounds provided herein are useful fortreating Trk-associated cancers in pediatric patients. For example, thecompounds provided herein can be used to treat infantile sarcoma,neuroblastoma, congenital mesoblastic nephroma, brain low-grade glioma,and pontine glioma.

In some embodiments, the compounds provided herein are useful fortreating a Trk-associated cancer in combination with one or moreadditional therapeutic agents or therapies that work by the same or adifferent mechanism of action.

In some embodiments, the additional therapeutic agent(s) is selectedfrom the group of: receptor tyrosine kinase-targeted therapeutic agents,including cabozantinib, crizotinib, erlotinib, gefitinib, imatinib,lapatinib, nilotinib, pazopanib, pertuzumab, regorafenib, sunitinib, andtrastuzumab.

In some embodiments, the additional therapeutic agent(s) is selectedfrom signal transduction pathway inhibitors, including, e.g.,Ras-Raf-MEK-ERK pathway inhibitors (e.g., sorafenib, trametinib, orvemurafenib), PI3K-Akt-mTOR-S6K pathway inhibitors (e.g., everolimus,rapamycin, perifosine, or temsirolimus) and modulators of the apoptosispathway (e.g., obataclax).

In some embodiments, the additional therapeutic agent(s) is selectedfrom the group of: cytotoxic chemotherapeutics, including, e.g., arsenictrioxide, bleomycin, cabazitaxel, capecitabine, carboplatin, cisplatin,cyclophosphamide, cytarabine, dacarbazine, daunorubicin, docetaxel,doxorubicin, etoposide, fluorouracil, gemcitabine, irinotecan,lomustine, methotrexate, mitomycin C, oxaliplatin, paclitaxel,pemetrexed, temozolomide, and vincristine.

In some embodiments, the additional therapeutic agent(s) is selectedfrom the group of angiogenesis-targeted therapies, including e.g.,aflibercept and bevacizumab.

In some embodiments, the additional therapeutic agent(s) is selectedfrom the group of immune-targeted agents, e.g., including aldesleukin,ipilimumab, lambrolizumab, nivolumab, and sipuleucel-T.

In some embodiments, the additional therapeutic agent(s) is selectedfrom agents active against the downstream Trk pathway, including, e.g.,NGF-targeted biopharmaceuticals, such as NGF antibodies and panTrkinhibitors.

In some embodiments, the additional therapeutic agent or therapy isradiotherapy, including, e.g., radioiodide therapy, external-beamradiation, and radium 223 therapy.

In some embodiments, the additional therapeutic agent(s) includes anyone of the above listed therapies or therapeutic agents which arestandards of care in cancers wherein the cancer has a dysregulation of aNTRK gene, a Trk protein, or expression or activity, or level of thesame.

Methods of detecting dysregulation of a NTRK gene, a Trk protein, orexpression or activity, or level of the same, include, e.g., detectionof NTRK gene translocations, e.g., using Fluorescent In SituHybridization (FISH) (e.g., as described in International ApplicationNos. PCT/US2013/061211 PCT/US2013/057495, which are incorporated hereinby reference).

In some embodiments, provided herein is a method of treating cancer(e.g., a Trk-associated cancer) in a patient, comprising administeringto said patient crystalline form (I-HS) or a compound of Formula I or asalt thereof, such as a hydrogen sulfate salt (e.g., see Example 14A ofU.S. Pat. No. 8,513,263) in combination with at least one additionaltherapy or therapeutic agent. In some embodiments, the at least oneadditional therapy or therapeutic agent is selected from radiotherapy(e.g., radioiodide therapy, external-beam radiation, or radium 223therapy), cytotoxic chemotherapeutics (e.g., arsenic trioxide,bleomycin, cabazitaxel, capecitabine, carboplatin, cisplatin,cyclophosphamide, cytarabine, dacarbazine, daunorubicin, docetaxel,doxorubicin, etoposide, fluorouracil, gemcitabine, irinotecan,lomustine, methotrexate, mitomycin C, oxaliplatin, paclitaxel,pemetrexed, temozolomide, or vincristine), tyrosine kinasetargeted-therapeutics (e.g., afatinib, cabozantinib, cetuximab,crizotinib, dabrafenib, erlotinib, gefitinib, imatinib, lapatinib,nilotinib, pazopanib, panitumumab, pertuzumab, regorafenib, sunitinib,or trastuzumab), apoptosis modulators and signal transduction inhibitors(e.g. everolimus, perifosine, rapamycin, sorafenib, temsirolimus,trametinib, or vemurafenib), immune-targeted therapies (e.g.,aldesleukin, interferon alfa-2b, ipilimumab, lambrolizumab, nivolumab,prednisone, or sipuleucel-T) and angiogenesis-targeted therapies (e.g.,aflibercept or bevacizumab), wherein the amount of a compound providedherein or a pharmaceutically acceptable salt thereof is, in combinationwith the additional therapy or therapeutic agent, is effective intreating said cancer.

In some embodiments, the additional therapeutic agent is a different Trkinhibitor. Non-limiting examples of other Trk inhibitors include a(R)-2-phenylpyrrolidine substituted imadazopyridazine, AZD6918,GNF-4256, GTx-186, GNF-5837, AZ623, AG-879, altiratinib, CT327, AR-772,AR-523, AR-786, AR-256, AR-618, AZ-23, AZD7451, cabozantinib, CEP-701,CEP-751, PHA-739358, dovitinib, entrectinib, PLX7486, Gö 6976, GW441756,MGCD516, ONO-5390556, PHA-848125AC, regorafenib, sorafenib, sunitinib,TSR-011, VM-902A, K252a, a 4-aminopyrazolylpyrimidine, and a substitutedpyrazolo[1,5-a]pyrimidine compound.

In some embodiments, the additional therapeutic agents include: receptortyrosine kinase-targeted therapeutic agents, such as afatinib,cabozantinib, cetuximab, crizotinib, dabrafenib, erlotinib, gefitinib,imatinib, lapatinib, lestaurtinib, nilotinib, pazopanib, panitumumab,pertuzumab, sunitinib, trastuzumab, AG 879, AZ-23, AZ623, Gö 6976,GNF-5837, GTx-186, GW 441756, MGCD516, RPI-1, RXDX101, and TSR-011;RET-targeted therapeutic agents, such as alectinib, apatinib,cabozantinib, dovitinib, lenvatinib, motesanib, nintedanib, ponatinib,regorafenib, sunitinib, sorafenib, vatalanib, vandetanib, AUY-922,BLU6864, DCC-2157, MGCD516, NVP-AST487, PZ-1, RXDX105, SPP86, TG101209,and XL-184; signal transduction pathway inhibitors, such asRas-Raf-MEK-ERK pathway inhibitors (e.g., binimetinib, selumetinib,encorafinib, sorafenib, trametinib, and vemurafenib), PI3K-Akt-mTOR-S6Kpathway inhibitors (e.g. everolimus, rapamycin, perifosine,temsirolimus), other kinase inhibitors, such as baricitinib, brigatinib,capmatinib, danusertib, ibrutinib, milciclib, quercetin, regorafenib,ruxolitinib, semaxanib, AP32788, BLU285, BLU554, INCB39110, INCB40093,INCB50465, INCB52793, INCB54828, MGCD265, NMS-088, NMS-1286937, PF477736, PLX3397, PLX7486, PLX8394, PLX9486, PRN1008, PRN1371, RXDX103,RXDX106, RXDX108, and TG101209; checkpoint inhibitors, such asipilimumab, tremelimumab, nivolumab, pidilizumab, MPDL3208A, MEDI4736,MSB0010718C, BMS-936559, BMS-956559, BMS-935559 (MDX-1105), AMP-224, andpembrolizumab; modulators of the apoptosis pathway (e.g. obataclax);cytotoxic chemotherapeutics, such as arsenic trioxide, bleomycin,cabazitaxel, capecitabine, carboplatin, cisplatin, cyclophosphamide,cytarabine, dacarbazine, daunorubicin, docetaxel, doxorubicin,etoposide, fluorouracil, gemcitabine, irinotecan, lomustine,methotrexate, mitomycin C, oxaliplatin, paclitaxel, pemetrexed,temozolomide, and vincristine; angiogenesis-targeted therapies, such asaflibercept and bevacizumab; immune-targeted agents, such asaldesleukin, interferon alfa-2b, ipilimumab, lambrolizumab, nivolumab,prednisone, sipuleucel-T; radiotherapy, such as radioiodide therapy,external-beam radiation, and radium 223 therapy.

Yet other additional therapeutic agents include RET inhibitors such asthose described, for example, in U.S. Pat, Nos. 8,299,057; 8,399,442;8,937,071; 9,006,256; and 9,035,063; U.S. Publication Nos. 2014/0121239;2011/0053934; 2011/0301157; 2010/0324065; 2009/0227556; 2009/0130229;2009/0099167; 2005/0209195; International Publication Nos. WO2014/184069; WO 2014/072220; WO 2012/053606; WO 2009/017838; WO2008/031551; WO 2007/136103; WO 2007/087245; WO 2007/057399; WO2005/051366; and WO 2005/044835; and J. Med.Chem. 2012, 55 (10),4872-4876.

These additional therapeutic agents may be administered with one or morecompounds provided herein as part of the same or separate dosage forms,via the same or different routes of administration, and on the same ordifferent administration schedules according to standard pharmaceuticalpractice known to one skilled in the art.

Also provided herein is (i) a pharmaceutical combination for treatingcancer (e.g., a Trk-associated cancer) in a patient in need thereof,which comprises (a) crystalline form (I-HS) or a compound of Formula Ior a salt thereof, such as a hydrogen sulfate salt (e.g., see Example14A of U.S. Pat. No. 8,513,263), (b) an additional therapeutic agent and(c) optionally at least one pharmaceutically acceptable carrier forsimultaneous, separate or sequential use for the treatment of a tumordisease, wherein the amounts of the compound or salt thereof and of theadditional therapeutic agent are together effective in treating saidcancer; (ii) a pharmaceutical composition comprising such a combination;(iii) the use of such a combination for the preparation of a medicamentfor the treatment of cancer (e.g., a Trk-associated cancer); and (iv) acommercial package or product comprising such a combination as acombined preparation for simultaneous, separate or sequential use; andto a method of treatment of cancer (e.g., Trk-associated cancer) in apatient in need thereof.

Also provided are methods of treating a subject identified or diagnosedas having a Trk-associated cancer (e.g., a subject that has beenidentified or diagnosed as having a Trk-associated cancer through theuse of a regulatory agency-approved, e.g., FDA-approved, kit foridentifying dysregulation of a NTRK gene, a Trk protein, or expressionor activity, or level of the same, in a subject or a biopsy sample fromthe subject) (e.g., any of the Trk-associated cancers described hereinor known in the art) that include administering the subject atherapeutically effective amount of crystalline form (I-HS) or acompound of Formula I or a salt thereof, such as a hydrogen sulfate salt(e.g., see Example 14A of U.S. Pat. No. 8,513,263). Also provided iscrystalline form (I-HS) or a compound of Formula I or a salt thereof,such as a hydrogen sulfate salt (e.g., see Example 14A of U.S. Pat. No.8,513,263) for use in treating a Trk-associated cancer in a subjectidentified or diagnosed as having a Trk-associated cancer (e.g., asubject that has been identified or diagnosed as having a Trk-associatedcancer through the use of a regulatory agency-approved, e.g.,FDA-approved, kit for identifying dysregulation of a NTRK gene, a Trkprotein, or expression or activity, or level of the same, in a subjector a biopsy sample from the subject) (e.g., any of the Trk-associatedcancers described herein or known in the art). Also provided is the useof crystalline form (I-HS) or a compound of Formula I or a salt thereof,such as a hydrogen sulfate salt (e.g., see Example 14A of U.S. Pat. No.8,513,263) for the manufacture of a medicament for treating aTrk-associated cancer in a subject identified or diagnosed as having aTrk- associated cancer (e.g., a subject that has been identified ordiagnosed as having a Trk-associated cancer through the use of aregulatory agency-approved, e.g., FDA-approved, kit for identifyingdysregulation of a NTRK gene, a Trk protein, or expression or activity,or level of the same, in a subject or a biopsy sample from the subject)(e.g., any of the Trk-associated cancers described herein or known inthe art).

Also provided are methods of treating a subject (e.g., a subjectsuspected of having a Trk-associated cancer, a subject presenting withone or more symptoms of a Trk-associated cancer, or a subject having anelevated risk of developing a Trk-associated cancer) that includeperforming an assay (e.g., an assay that utilizes next generationsequencing, immunohistochemistry, or break apart FISH analysis) (e.g.,using a regulatory agency-approved, e.g., FDA-approved, kit) on a sampleobtained from the subject to determine whether the subject hasdysregulation of a NTRK gene, a Trk protein, or expression or activity,or level of the same, and administering (e.g., specifically orselectively administering) a therapeutically effective amount ofcrystalline form (I-HS) or a compound of Formula I or a salt thereof,such as a hydrogen sulfate salt (e.g., see Example 14A of U.S. Pat. No.8,513,263) to a subject determined to have dysregulation of a NTRK gene,a Trk protein, or expression or activity, or levels of the same.Additional assays, non-limiting assays that may be used in these methodsare described herein. Additional assays are also known in the art. Alsoprovided is use of crystalline form (I-HS) or a compound of Formula I ora salt thereof, such as a hydrogen sulfate salt (e.g., see Example 14Aof U.S. Pat. No. 8,513,263) for use in treating a Trk-associated cancerin a subject identified or diagnosed as having a Trk-associated cancerthrough a step of performing an assay (e.g., an in vitro assay) (e.g.,an assay that utilizes next generation sequencing, immunohistochemistry,or break apart FISH analysis) (e.g., using a regulatory agency-approved,e.g., FDA-approved, kit) on a sample obtained from the subject todetermine whether the subject has dysregulation of a NTRK gene, a Trkprotein, or expression or activity, or level of the same, where thepresence of dysregulation of a NTRK gene, a Trk protein, or expressionor activity, or level of the same, identifies that the subject has aTrk-associated cancer. Also provided is the use of crystalline form(I-HS) or a compound of Formula I or a salt thereof, such as a hydrogensulfate salt (e.g., see Example 14A of U.S. Pat. No. 8,513,263) for themanufacture of a medicament for treating a Trk-associated cancer in asubject identified or diagnosed as having a Trk-associated cancerthrough a step of performing an assay (e.g., an in vitro assay) (e.g.,an assay that utilizes next generation sequencing, immunohistochemistry,or break apart FISH analysis) (e.g., using a regulatory agency-approved,e.g., FDA-approved, kit) on a sample obtained from the subject todetermine whether the subject has dysregulation of a NTRK gene, a Trkprotein, or expression or activity, or level of the same, where thepresence of dysregulation of a NTRK gene, a Trk protein, or expressionor activity, or level of the same, identifies that the subject has aTrk-associated cancer. Some embodiments of any of the methods or usesdescribed herein further include recording in the subject's clinicalrecord (e.g., a computer readable medium) that the subject determined tohave dysregulation of a NTRK gene, a Trk protein, or expression oractivity, or level of the same, through the performance of the assay,should be administered a crystalline form (I-HS) or a compound ofFormula I or a salt thereof, such as a hydrogen sulfate salt (e.g., seeExample 14A of U.S. Pat. No. 8,513,263).

In some embodiments of any of the methods or uses described herein, thesubject has been identified or diagnosed as having a cancer withdysregulation of a NTRK gene, a Trk protein, or expression or activity,or level of the same (e.g., as determined using a regulatoryagency-approved, e.g., FDA-approved, assay or kit). In some embodimentsof any of the methods or uses described herein, the subject has a tumorthat is positive for dysregulation of a NTRK gene, a Trk protein, orexpression or activity, or level of the same (e.g., as determined usinga regulatory agency-approved assay or kit). In some embodiments of anyof the methods or uses described herein, the subject can be a subjectwith a tumor(s) that is positive for dysregulation of a NTRK gene, a Trkprotein, or expression or activity, or level of the same (e.g.,identified as positive using a regulatory agency-approved, e.g.,FDA-approved, assay or kit). In some embodiments of any of the methodsor uses described herein, the subject can be a subject whose tumors havedysregulation of a NTRK gene, a Trk protein, or expression or activity,or a level of the same (e.g., where the tumor is identified as suchusing a regulatory agency-approved, e.g., FDA-approved, kit or assay).In some embodiments of any of the methods or uses described herein, thesubject is suspected of having a Trk-associated cancer. In someembodiments of any of the methods or uses described herein, the subjecthas a clinical record indicating that the subject has a tumor that hasdysregulation of a NTRK gene, a Trk protein, or expression or activity,or level of the same (and optionally the clinical record indicates thatthe subject should be treated with any of the compositions providedherein).

Also provided are methods of treating a subject that includeadministering a therapeutically effective amount of crystalline form(I-HS) or a compound of Formula I or a salt thereof, such as a hydrogensulfate salt (e.g., see Example 14A of U.S. Pat. No. 8,513,263) to asubject having a clinical record that indicates that the subject hasdysregulation of a NTRK gene, a Trk protein, or expression or activity,or level of the same. Also provided is the use of crystalline form(I-HS) or a compound of Formula I or a salt thereof, such as a hydrogensulfate salt (e.g., see Example 14A of U.S. Pat. No. 8,513,263) for themanufacture of a medicament for treating a Trk-associated cancer in asubject having a clinical record that indicates that the subject hasdysregulation of a NTRK gene, a Trk protein, or expression or activity,or level of the same. Also provided is the use of crystalline form(I-HS) or a compound of Formula I or a salt thereof, such as a hydrogensulfate salt (e.g., see Example 14A of U.S. Pat. No. 8,513,263 for themanufacture of a medicament for treating a Trk-associated cancer in asubject having a clinical record that indicates that the subject hasdysregulation of a NTRK gene, a Trk protein, or expression or activity,or level of the same. Some embodiments of these methods and uses canfurther include: a step of performing an assay (e.g., an in vitro assay)(e.g., an assay that utilizes next generation sequencing,immunohistochemistry, or break apart FISH analysis) (e.g., using aregulatory agency-approved, e.g., FDA-approved, kit) on a sampleobtained from the subject to determine whether the subject hasdysregulation of a NTRK gene, a Trk protein, or expression or activity,or level of the same, and recording information in a subject's clinicalfile (e.g., a computer-readable medium) that the subject has beenidentified to have dysregulation of a NTRK gene, a Trk protein, orexpression or activity, or level of the same.

Also provided are methods (e.g., in vitro methods) of selecting atreatment for a subject that include selecting a treatment includingadministration of a therapeutically effective amount of crystalline form(I-HS) or a compound of Formula I or a salt thereof, such as a hydrogensulfate salt (e.g., see Example 14A of U.S. Pat. No. 8,513,263) for asubject identified or diagnosed as having a Trk-associated cancer (e.g.,a subject that has been identified or diagnosed as having aTrk-associated cancer through the use of a regulatory agency-approved,e.g., FDA-approved, kit for identifying dysregulation of a NTRK gene, aTrk protein, or expression or activity, or level of the same, in asubject or a biopsy sample from the subject) (e.g., any of theTrk-associated cancers described herein or known in the art). Someembodiments can further include administering the selected treatment tothe subject identified or diagnosed as having a Trk-associated cancer.Some embodiments can further include a step of performing an assay(e.g., an in vitro assay) (e.g., an assay that utilizes next generationsequencing, immunohistochemistry, or break apart FISH analysis) (e.g.,using a regulatory agency-approved, e.g., FDA-approved, kit) on a sampleobtained from the subject to determine whether the subject hasdysregulation of a NTRK gene, a Trk protein, or expression or activity,or level of the same, and identifying or diagnosing a subject determinedto have dysregulation of a NTRK gene, a Trk protein, or expression oractivity, or level of the same, as having a Trk-associated cancer.

Also provided are methods of selecting a treatment for a subject thatinclude administration of a therapeutically effective amount ofcrystalline form (I-HS) or a compound of Formula I or a salt thereof,such as a hydrogen sulfate salt (e.g., see Example 14A of U.S. Pat. No.8,513,263), wherein the methods include a step of performing an assay(e.g., an in vitro assay) (e.g., an assay that utilizes next generationsequencing, immunohistochemistry, or break apart FISH analysis) (e.g.,using a regulatory agency-approved, e.g., FDA-approved, kit) on a sampleobtained from the subject to determine whether the subject hasdysregulation of a NTRK gene, a Trk protein, or expression or activity,or level of the same, and identifying or diagnosing a subject determinedto have dysregulation of a NTRK gene, a Trk protein, or expression oractivity, or level of the same, as having a Trk-associated cancer, andselecting a therapeutic treatment including administration of atherapeutically effective amount of crystalline form (I-HS) or acompound of Formula I or a salt thereof, such as a hydrogen sulfate salt(e.g., see Example 14A of U.S. Pat. No. 8,513,263) for the subjectidentified or diagnosed as having a Trk-associated cancer. Someembodiments further include administering the selected treatment to thesubject identified or diagnosed as having a Trk-associated cancer.

Also provided are methods of selecting a subject for treatment includingadministration of a therapeutically effective amount of crystalline form(I-HS) or a compound of Formula I or a salt thereof, such as a hydrogensulfate salt (e.g., see Example 14A of U.S. Pat. No. 8,513,263) thatinclude selecting, identifying, or diagnosing a subject having aTrk-associated cancer, and selecting the subject for treatment includingadministration of a therapeutically effective amount of crystalline form(I-HS) or a compound of Formula I or a salt thereof, such as a hydrogensulfate salt (e.g., see Example 14A of U.S. Pat. No. 8,513,263). In someembodiments, identifying or diagnosing a subject as having aTrk-associated cancer can include a step of performing an assay (e.g.,an in vitro assay) (e.g., an assay that utilizes next generationsequencing, immunohistochemistry, or break apart FISH analysis) (e.g.,using a regulatory agency-approved, e.g., FDA-approved, kit) on a sampleobtained from the subject to determine whether the subject hasdysregulation of a NTRK gene, a Trk protein, or expression or activity,or level of the same, and identifying or diagnosing a subject determinedto have dysregulation of a NTRK gene, a Trk protein, or expression oractivity, or level of the same, as having a Trk-associated cancer. Insome embodiments, the selecting a treatment can be used as part of aclinical study that includes administration of various treatments of anAlk-associated cancer.

In some embodiments of any of the methods or uses described herein, anassay used determine whether the subject has dysregulation of a NTRKgene, a Trk protein, or expression or activity, or level of the same,using a sample (e.g., a biological sample or a biopsy sample (e.g., aparaffin-embedded biopsy sample) from a subject (e.g., a subjectsuspected of having a Trk-associated cancer, a subject having one ormore symptoms of a Trk-associated cancer, and/or a subject that has anincreased risk of developing a Trk-associated cancer) can include, forexample, next generation sequencing, immunohistochemistry, fluorescencemicroscopy, break apart FISH analysis, Southern blotting, Westernblotting, FACS analysis, Northern blotting, and PCR-based amplification(e.g., RT-PCR). As is well-known in the art, the assays are typicallyperformed, e.g., with at least one labelled nucleic acid probe or atleast one labelled antibody or antigen-binding fragment thereof. Assayscan utilize other detection methods known in the art for detectingdysregulation of a NTRK gene, a Trk protein, or expression or activity,or levels of the same (see, e.g., the references cited herein).

In some embodiments, crystalline form (I-HS) or a compound of Formula Ior a salt thereof, such as a hydrogen sulfate salt (e.g., see Example14A of U.S. Pat. No. 8,513,263) is useful for treating chronic and acutepain, including pain associated with cancer, surgery, and bone fracture.Crystalline form (I-HS) or a compound of Formula I or a salt thereof,such as a hydrogen sulfate salt (e.g., see Example 14A of U.S. Pat. No.8,513,263) may be useful in the treatment of multiple types of painincluding inflammatory pain, neuropathic pain, and pain associated withcancer, surgery, and bone fracture. Crystalline form (I-HS) or acompound of Formula I or a salt thereof, such as a hydrogen sulfate salt(e.g., see Example 14A of U.S. Pat. No. 8,513,263) are also useful fortreating cancers including neuroblastoma, ovarian, pancreatic andcolorectal cancer. Crystalline form (I-HS) or a compound of Formula I ora salt thereof, such as a hydrogen sulfate salt (e.g., see Example 14Aof U.S. Pat. No. 8,513,263) is also useful for treating inflammation andcertain infectious diseases. In addition, crystalline form (I-HS) or acompound of Formula I or a salt thereof, such as a hydrogen sulfate salt(e.g., see Example 14A of U.S. Pat. No. 8,513,263) may also be used totreat interstitial cystitis (IC), painful bladder syndrome (PBS),urinary incontinence, asthma, anorexia, atopic dermatitis, andpsoriasis. Crystalline form (I-HS) or a compound of Formula I or a saltthereof, such as a hydrogen sulfate salt (e.g., see Example 14A of U.S.Pat. No. 8,513,263) may also be used to treat demyelination anddysmyelination by promoting myelination, neuronal survival, andoligodendrocyte differentiation via blocking Sp35-TrkA interaction.Crystalline form (I-HS) or a compound of Formula I or a salt thereof,such as a hydrogen sulfate salt (e.g., see Example 14A of U.S. Pat. No.8,513,263) may be useful in the treatment of multiple types of painincluding inflammatory pain, neuropathic pain, surgical pain and painassociated with cancer. Crystalline form (I-HS) or a compound of FormulaI or a salt thereof, such as a hydrogen sulfate salt (e.g., see Example14A of U.S. Pat. No. 8,513,263) may be useful in the treatment ofbone-related diseases (such as those involving bone resorption).Examples of bone-related diseases include metastatic bone disease,treatment-induced bone loss, osteoporosis, rheumatoid arthritis,ankylosing spondylitis, Paget's disease, and periodontal disease. Theosteoporosis may be attributed to (1) menopause in women, (2) aging inmen or women, (3) suboptimal bone growth during childhood andadolescence that resulted in failure to reach peak bone mass, and/or (4)bone loss secondary to other disease conditions, eating disorders,medications and/or medical treatments. Other osteolytic diseases thatcan be treated according to the methods provided herein are morelocalized. A particular example is metastatic tumor-induced osteolysis.In this condition, bone cancers or bone metastases induce localizedosteolysis that causes pain, bone weakness and fractures. Such localizedosteolysis also permits tumors to grow larger by creating more space forthem in the bone and releasing growth factors from the bone matrix.Cancers presently known to cause tumor-induced osteolysis includehematological malignancies (e.g., myeloma and lymphoma) and solid tumors(e.g., breast, prostate, lung, renal and thyroid), all of which thepresent disclosure contemplates treating. As used herein, the termtreatment includes prophylaxis as well as treatment of an existingcondition.

Accordingly, also provided herein is a method of treating diseases ormedical conditions in a subject in need thereof, wherein said disease orcondition is treatable with an inhibitor of TrkA and/or TrkB (e.g., aTrk-associated cancer), comprising administering to said subjectcrystalline form (I-HS) or a compound of Formula I or a salt thereof,such as a hydrogen sulfate salt (e.g., see Example 14A of U.S. Pat. No.8,513,263 in an amount effective to treat or prevent said disorder. In aparticular embodiment, provided herein is a method of treating pain,cancer, inflammation, neurodegenerative disease or Trypanosoma cruziinfection in a mammal, which comprises administering to said mammal atherapeutically effective amount of crystalline form (I-HS) or acompound of Formula I or a salt thereof, such as a hydrogen sulfate salt(e.g., see Example 14A of U.S. Pat. No. 8,513,263). In anotherembodiment, provided herein is a method of treating osteolytic diseasein a mammal, which comprises administering to said subject in needthereof a therapeutically effective amount of crystalline form (I-HS) ora compound of Formula I or a salt thereof, such as a hydrogen sulfatesalt (e.g., see Example 14A of U.S. Pat. No. 8,513,263).

Crystalline form (I-HS) or a compound of Formula I or a salt thereof,such as a hydrogen sulfate salt (e.g., see Example 14A of U.S. Pat. No.8,513,263) can be used in combination with one or more additional drugsthat work by the same or a different mechanism of action. Such conjointtreatment may be achieved by way of the simultaneous, sequential orseparate administration of the individual components of the treatment.Examples include anti-inflammatory compounds, steroids (e.g.,dexamethasone, cortisone and fluticasone), analgesics such as NSAIDs(e.g., aspirin, ibuprofen, indomethacin, and ketoprofen), and opioids(such as morphine), and chemotherapeutic agents.

In the field of medical oncology it is normal practice to use acombination of different forms of treatment to treat each patient withcancer. In medical oncology the other component(s) of such conjointtreatment in addition to compositions provided herein may be, forexample, surgery, radiotherapy, chemotherapy, signal transductioninhibitors and/or monoclonoal antibodies.

Accordingly, crystalline form (I-HS) may be administered in combinationwith one or more agents selected from mitotic inhibitors, alkylatingagents, anti-metabolites, antisense DNA or RNA, intercalatingantibiotics, growth factor inhibitors, signal transduction inhibitors,cell cycle inhibitors, enzyme inhibitors, retinoid receptor modulators,proteasome inhibitors, topoisomerase inhibitors, biological responsemodifiers, anti-hormones, angiogenesis inhibitors, cytostatic agentsanti-androgens, targeted antibodies, HMG-CoA reductase inhibitors, andprenyl-protein transferase inhibitors.

Where the compound disclosed herein has at least one chiral center, thecompounds may accordingly exist as enantiomers. Where the compoundspossess two chiral centers, the compounds may additionally exist asdiastereomers. That is, the compound of Formula I, in addition to havingthe desired configuration designated by the nomenclature“(S)—N-(5((R)-2-(2,5-difluorophenyl)-pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehydrogen sulfate” (hereinafter referred to as the (S,R) isomer), it mayalso be present in minor amounts as the isomer(R)—N-(5((R)-2-(2,5-difluorophenyl)-pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehydrogen sulfate (hereinafter referred to as the (R,R) isomer) and/ormay also be present in minor amounts as the(S)—N-(5((S)-2-(2,5-difluorophenyl)-pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehydrogen sulfate (hereinafter referred to as the (S,S) isomer), and/ormay be present in minor amounts as the isomer(R)—N-(5((S)-2-(2,5-difluorophenyl)-pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehydrogen sulfate” (hereinafter referred to as the (R,S) isomer). It isto be understood that all such isomers and mixtures thereof areencompassed within the scope of the present invention. Preferably,wherein the compound is present as the (S,R) isomer, the (S,R) isomer ispresent at an excess of greater than or equal to about 80%, morepreferably at an excess of greater than or equal to about 90%, morepreferably still at an excess of greater than or equal to about 95%,more preferably still at an excess of greater than or equal to about98%, more preferably at an excess of greater than or equal to about 99%.

It will be appreciated that crystalline form (I-HS) contains two centersof asymmetry and may therefore be prepared and isolated in a mixture ofisomers such as a racemic or diastereomeric mixture, or in anenantiomerically pure form. Where stereochemistry is specified by asolid wedge or dashed line representing a particular configuration, thenthat stereoisomer is so specified and defined.

As used herein, unless otherwise noted, the term “isolated form” shallmean that the compound is present in a form which is separate from anysolid mixture with another compound(s), solvent system or biologicalenvironment. In some embodiments, the crystalline form (I-HS) is presentas an isolated form.

As used herein, unless otherwise noted, the term “substantially pureform” shall mean that the mole percent of impurities in the isolatedcompound or crystalline form is less than about 5 mole percent,preferably less than about 2 mole percent, more preferably, less thanabout 0.5 mole percent, most preferably, less than about 0.1 molepercent. In some embodiments, the crystalline form (I-HS) is present asa substantially pure form.

As used herein, unless otherwise noted, the term “substantially free ofother amorphous, polymorph or crystalline form(s)” when used todescribed crystalline form (I-HS) shall mean that mole percent of otheramorphous, polymorph or crystalline form(s) of the isolated base ofcrystalline form (I-HS) is less than about 5 mole percent, preferablyless than about 2 mole percent, more preferably, less than about 0.5mole percent, most preferably less than about 0.1 mole percent. In someembodiments, the crystalline form (I-HS) is present as a formsubstantially free of other amorphous, polymorph or crystalline form(s).

The terms “polymorph” and “polymorphic form” refer to differentcrystalline forms of a single compound. That is, polymorphs are distinctsolids sharing the same molecular formula, yet each polymorph may havedistinct solid state physical properties. Therefore, a single compoundmay give rise to a variety of polymorphic forms where each form hasdifferent and distinct solid state physical properties, such asdifferent solubility profiles, dissolution rates, melting pointtemperatures, flowability, and/or different X-ray diffraction peaks. Thedifferences in physical properties may affect pharmaceutical parameterssuch as storage stability, compressibility and density (which can beimportant in formulation and product manufacturing), and dissolutionrate (which can be an important factor in bioavailability). Techniquesfor characterizing polymorphic forms include, but are not limited to,X-ray powder diffractometry (XRPD), differential scanning calorimetry(DSC), thermal gravimetric analysis (TGA), single-crystal X-raydiffractometry (XRD), vibrational spectroscopy, e.g., infrared (IR) andRaman spectroscopy, solid-state and solution nuclear magnetic resonance(NMR) spectroscopy, optical microscopy, hot stage optical microscopy,scanning electron microscopy (SEM), electron crystallography andquantitative analysis, particle size analysis (PSA), surface areaanalysis, solubility measurements, dissolution measurements, elementalanalysis and Karl Fischer analysis.

The term “amorphous” means a solid in a solid state that is anon-crystalline state. Amorphous solids are disordered arrangements ofmolecules and therefore possess no distinguishable crystal lattice orunit cell and consequently have no definable long range ordering. Thesolid state form of a solid may be determined by polarized lightmicroscopy, X-ray powder diffraction (“XRPD”), differential scanningcalorimetry (“DSC”), or other standard techniques known to those ofskill in the art.

As used herein, unless otherwise noted, the terms “treating,”“treatment,” and the like, shall include the management and care of asubject or patient (preferably mammal, more preferably human) for thepurpose of combating a disease, condition, or disorder and includes theadministration of a disclosed compound to alleviate the symptoms orcomplications, or reduce the rate of progression of the disease,condition, or disorder.

As used herein, unless otherwise noted, the term “prevention” shallinclude (a) reduction in the frequency of one or more symptoms; (b)reduction in the severity of one or more symptoms; (c) the delay oravoidance of the development of additional symptoms; and/or (d) delay oravoidance of the development of the disorder or condition.

As used herein, the term “Trk-associated cancer” shall be defined toinclude cancers associated with or having dysregulation of a NTRK gene,a Trk protein, or expression or activity, or level of the same (e.g.,any of types of dysregulation of a NTRK gene, a Trk protein, orexpression or activity, or level of the same, described herein).Non-limiting examples of a Trk-associated cancer are described herein.

As used herein, the term “pain” shall be defined to include acute,chronic, inflammatory and neuropathic pain, including diabeticneuropathy. Further, the pain may be centrally mediated, peripherallymediated, caused by structural tissue injury, caused by soft tissueinjury or caused by progressive disease. Any centrally mediated,peripherally mediated, structural tissue injury, soft tissue injury orprogressive disease related pain may be acute or chronic.

As used herein, unless otherwise noted, pain shall include inflammatorypain, centrally mediated pain, peripherally mediated pain, visceralpain, structural related pain, cancer pain, soft tissue injury relatedpain, progressive disease related pain, neuropathic pain, acute painfrom acute injury, acute pain from trauma, acute pain from surgery,headache, dental pain, back pain (preferably lower back pain), chronicpain from neuropathic conditions and chronic pain from post-strokeconditions.

Some embodiments include methods for the treatment of pain, wherein thepain is acute pain. Some embodiments include methods for the treatmentof pain, wherein the pain is chronic pain. Some embodiments includemethods for the treatment of pain, wherein the pain is neuropathic pain,including diabetic neuropathy. Some embodiments include methods for thetreatment of pain, wherein the pain is inflammatory pain.

In some embodiments, the pain is selected from the group consisting ofosteoarthritis, rheumatoid arthritis, fibromyalgia, headache, toothache,burn, sunburn, animal bite (such as dog bite, cat bite, snake bite,spider bite, insect sting, and the like), neurogenic bladder, benignprostatic hypertrophy, interstitial cystitis, rhinitis, contactdermatitis/hypersensitivity, itch, eczema, pharyngitis, mucositis,enteritis, cellulites, causalgia, sciatic neuritis, mandibular jointneuralgia, peripheral neuritis, polyneuritis, stump pain, phantom limbpain, post-operative ileus, cholecystitis, postmastectomy pain syndrome,oral neuropathic pain, Charcot's pain, reflex sympathetic dystrophy,Guillain-Barre syndrome, meralgia paresthetica, burning-mouth syndrome,post-herpetic neuralgia, trigeminal neuralgia, peripheral neuropathy,bilateral peripheral neuropathy, diabetic neuropathy, postherpeticneuralgia, trigeminal neuralgia, optic neuritis, postfebrile neuritis,migrating neuritis, segmental neuritis, Gombault's neuritis, neuronitis,cervicobrachial neuralgia, cranial neuralgia, geniculate neuralgia,glossopharyngial neuralgia, migrainous neuralgia, idiopathic neuralgia,intercostals neuralgia, mammary neuralgia, Morton's neuralgia,nasociliary neuralgia, occipital neuralgia, red neuralgia, Sluder'sneuralgia, splenopalatine neuralgia, supraorbital neuralgia, vidianneuralgia, inflammatory bowel disease, irritable bowel syndrome, labor,childbirth, menstrual cramps, cancer, back pain, lower back pain andcarpal tunnel syndrome pain.

Acute pain includes pain caused by acute injury, trauma, illness orsurgery (for example, open-chest surgery (including open-heart or bypasssurgery)). Acute pain also includes, and is not limited to, headache,post-operative pain, kidney stone pain, gallbladder pain, gallstonepain, obstetric pain, rheumatological pain, dental pain or pain causedby sports-medicine injuries, carpal tunnel syndrome, burns,musculoskeletal sprains and strains, musculotendinous strain,cervicobrachial pain syndromes, dyspepsia, gastric ulcer, duodenalulcer, dysmenorrhea or endometriosis.

Chronic pain includes pain caused by an inflammatory condition,osteoarthritis, rheumatoid arthritis or as sequela to disease, acuteinjury or trauma. Chronic pain also includes, and is not limited to,headache, upper back pain or lower back pain (selected from back painresulting from systematic, regional or primary spine disease (selectedfrom radiculopathy)), bone pain (selected from bone pain due toosteoarthritis, osteoporosis, bone metastases or unknown reasons),pelvic pain, spinal cord injury-associated pain, cardiac chest pain,non-cardiac chest pain, central post-stroke pain, myofascial pain,cancer pain, AIDS pain, sickle cell pain, geriatric pain or pain causedby headache, migraine, trigeminal neuralgia, temporomandibular jointsyndrome, fibromyalgia syndrome, osteoarthritis, rheumatoid arthritis,gout, fibrositis or thoracic outlet syndromes.

Neuropathic pain includes pain resulting from chronic or debilitatingconditions or disorders. The chronic or debilitating conditions ordisorders which can lead to neuropathic pain include, but are notlimited to, painful diabetic peripheral neuropathy, post-herpeticneuralgia, trigeminal neuralgia, post-stroke pain, multiplesclerosis-associated pain, neuropathies-associated pain such as inidiopathic or post-traumatic neuropathy and mononeuritis, HIV-associatedneuropathic pain, cancer-associated neuropathic pain, carpaltunnel-associated neuropathic pain, spinal cord injury-associated pain,complex regional pain syndrome, fibromyalgia-associated neuropathicpain, lumbar and cervical pain, reflex sympathic dystrophy, phantom limbsyndrome and other chronic and debilitating condition-associated painsyndromes.

“Acute neurodegenerative disorders or diseases” include, but are notlimited to, various types of acute neurodegenerative disordersassociated with neuron death or damage including cerebrovascularinsufficiency, focal brain trauma, diffuse brain damage, and spinal cordinjury, that is, cerebral ischemia or infarction including embolicocclusion and thrombotic occlusion, reperfusion following acuteischemia, perinatal hypoxic-ischemic injury, cardiac arrest, as well asintracranial hemorrhage of any type (including, but not limited to,epidural, subdural, subarachnoid and intracerebral), and intracranialand intravertebral lesions (including, but not limited to, contusion,penetration, shear, compression and laceration), and whiplash shakeninfant syndrome. In some embodiments, the acute neurodegenerativedisorder is a result of stroke, acute ischemic injury, head injury orspinal injury.

“Chronic neurodegenerative disorders or diseases” include, but are notlimited to, Alzheimer's disease, Pick's disease, diffuse Lewy bodydisease, progressive supranuclear palsy (Steel-Richardson syndrome),multisystem degeneration (Shy-Drager syndrome), chronic epilepticconditions associated with neurodegeneration, motor neuron diseasesincluding amyotrophic lateral sclerosis, degenerative ataxias, corticalbasal degeneration, ALS-Parkinson's-Dementia complex of Guam, subacutesclerosing panencephalitis, Huntington's disease, Parkinson's disease,synucleinopathies (including multiple system atrophy), primaryprogressive aphasia, striatonigral degeneration, Machado-Josephdisease/spinocerebellar ataxia type 3 and olivopontocerebellardegenerations, Gilles De La Tourette's disease, bulbar and pseudobulbarpalsy, spinal and spinobulbar muscular atrophy (Kennedy's disease),multiple sclerosis, primary lateral sclerosis, familial spasticparaplegia, Werdnig-Hoffmann disease, Kugelberg-Welander disease,Tay-Sach's disease, Sandhoff disease, familial spastic disease,Wohlfart-Kugelberg-Welander disease, spastic paraparesis, progressivemultifocal leukoencephalopathy, familial dysautonomia (Riley-Daysyndrome), and prion diseases (including, but not limited toCreutzfeldt-Jakob, Gerstmann-Straussler-Scheinker disease, Kuru andfatal familial insomnia). In some embodiments, the chronicneurodegenerative disorder is selected from Alzheimer's disease,Parkinson's disease, multiple sclerosis or cerebral palsy.

The term “subject” as used herein, refers to an animal, preferably amammal, most preferably a human, who has been the object of treatment,observation or experiment. In some embodiments, the subject hasexperienced and/or exhibited at least one symptom of the disease ordisorder to be treated and/or prevented. In some embodiments, a patientis a pediatric patient (i.e. a patient under the age of 21 years at thetime of diagnosis or treatment). The term “pediatric” can be furtherdivided into various subpopulations including: neonates (from birththrough the first 28 days of life); infants (29 days of age to less thantwo years of age); children (two years of age to less than 12 years ofage); and adolescents (12 years of age through 21 years of age (up to,but not including, the twenty-second birthday)).

In some embodiments, the subject has been identified or diagnosed ashaving a cancer with dysregulation of a NTRK gene, a Trk protein, orexpression or activity, or level of the same (e.g., as determined usinga regulatory agency-approved, e.g., FDA-approved, assay or kit). In someembodiments, the subject has a tumor that is positive for dysregulationof a NTRK gene, a Trk protein, or expression or activity, or level ofthe same (e.g., as determined using a regulatory agency-approved assayor kit). The subject can be a subject with a tumor(s) that is positivefor dysregulation of a NTRK gene, a Trk protein, or expression oractivity, or level of the same (e.g., identified as positive using aregulatory agency-approved, e.g., FDA-approved, assay or kit). Thesubject can be a subject whose tumors have dysregulation of a NTRK gene,a Trk protein, or expression or activity, or a level of the same (e.g.,where the tumor is identified as such using a regulatoryagency-approved, e.g., FDA-approved, kit or assay). In some embodiments,the subject is suspected of having a Trk-associated cancer. In someembodiments, the subject has a clinical record indicating that thesubject has a tumor that has dysregulation of a NTRK gene, a Trkprotein, or expression or activity, or level of the same (and optionallythe clinical record indicates that the subject should be treated withany of the compositions provided herein).

The term “Trk” or “Trk protein” includes any of the Trk proteinsdescribed herein (e.g., a TrkA, a TrkB, or a TrkC protein).

The term “NTRK gene” includes any of the NTRK genes described herein(e.g., a NTRK1, a NTRK2, or a NTRK3 gene).

The term “wildtype” or “wild-type” describes a nucleic acid (e.g., aNTRK gene or a Trk mRNA) or protein (e.g., a Trk protein) that is foundin a subject that does not have a Trk-associated cancer (and optionallyalso does not have an increased risk of developing a Trk-associatedcancer or condition and/or is not suspected of having a Trk-associatedcancer or condition) or is found in a cell or tissue from a subject thatdoes not have a Trk-associated cancer or condition (and optionally alsodoes not have an increased risk of developing a Trk-associated cancer orcondition and/or is not suspected of having a Trk-associated cancer orcondition).

The term “regulatory agency” is a country's agency for the approval ofthe medical use of pharmaceutical agents with the country. For example,a non-limiting example of a regulatory agency is the U.S. Food and DrugAdministration (FDA).

The phrase “dysregulation of a NTRK gene, a Trk protein, or expressionor activity, or level of the same” is a genetic mutation (e.g., a NTRKgene translocation that results in the expression of a fusion protein, adeletion in a NTRK gene that results in the expression of a Trk proteinthat includes a deletion of at least one amino acid as compared to thewild-type Trk protein, or a mutation in a NTRK gene that results in theexpression of a Trk protein with one or more point mutations, analternative spliced version of a Trk mRNA that results in a Trk proteinthat results in the deletion of at least one amino acid in the Trkprotein as compared to the wild-type Trk protein), or a NTRK geneduplication that results in overexpression of a Trk protein) or anautocrine activity resulting from the overexpression of a NTRK gene acell, that results in a pathogenic increase in the activity of a kinasedomain of a Trk protein (e.g., a constitutively active kinase domain ofa Trk protein) in a cell. For example, a dysregulation of a NTRK gene, aTrk protein, or expression or activity, or level of the same, can be amutation in a NTRK1, NTRK2, or NTRK3 gene that encodes a Trk proteinthat is constitutively active or has increased activity as compared to aprotein encoded by a NTRK1, NTRK2, or NTRK3 gene that does not includethe mutation. For example, a dysregulation of a NTRK gene, a Trkprotein, or expression or activity, or level of the same, can be theresult of a gene translocation which results in the expression of afusion protein that contains a first portion of TrkA, TrkB, or TrkC thatincludes a functional kinase domain, and a second portion of a partnerprotein (i.e., that is not TrkA, TrkB, or TrkC). A gene encoding afusion protein can include, e.g., the following exons of a wild-typeNTRK1 gene: exons 10-19, exons 12-19, exons 12-19, exons 13-19, exons14-19, or exons 15-19. A gene encoding a fusion protein can include,e.g., the following exons of a wild-type NTRK2 gene: exons 12-21, exons13-21, exons 15-21, exons 16-21, or exons 17-21. A gene encoding afusion protein can include, e.g., the following exons of a wild-typeNTRK3 gene: exons 17-22 or exons 16-22. Non-limiting examples of fusionproteins that are a result of a NTRK gene translocation are described inTables 1, 3, and 4.

A dysregulation of a NTRK gene, a Trk protein, or expression oractivity, or level of the same, can, e.g., include a mutation(s) in aNTRK1, NTRK2, or NTRK3 gene that results in a TrkA, TrkB, or TrkCcontaining at least one (e.g., two, three, four, or five) pointmutations (e.g., one of more of the point mutations listed in Table 6).A dysregulation of a NTRK gene, a Trk protein, or expression oractivity, or level of the same, can, e.g., include a mutation in a NTRK2gene that results in a TrkB protein including a point mutation of V673M.A dysregulation of a NTRK gene, a Trk protein, or expression oractivity, or level of the same, can, e.g., include a mutation in a NTRK3gene that results in a TrkC protein including a point mutation of H677Y.

A dysregulation of a NTRK gene, a Trk protein, or expression oractivity, or level of the same, can be a mutation in a NTRK1, NTRK2, orNTRK3 gene that results in a deletion of one or more contiguous aminoacids (e.g., at least two, at least three, at least four, at least 5, atleast 6, at least 7, at least 8, at least 9, at least 10, at least 15,at least 20, at least 30, at least 40, at least 50, at least 60, atleast 70, at least 80, at least 90, at least 100, at least 110, at least120, at least 130, at least 140, at least 150, at least 160, at least170, at least 180, at least 190, at least 200, at least 210, at least220, at least 230, at least 240, at least 250, at least 260, at least270, at least 280, at least 290, at least 300, at least 310, at least320, at least 330, at least 340, at least 350, at least 360, at least370, at least 380, at least 390, or at least 400 amino acids) in theTrkA, TrkB, or TrkC protein (except for the deletion of amino acids inthe kinase domain of TrkA, TrkB, or TrkC that would result ininactivation of the kinase domain). In some embodiments, dysregulationof a NTRK gene, a Trk protein, or expression or activity, or level ofthe same, can include a deletion in a NTRK1 gene that results in a TrkAprotein that lacks the NGF-binding site or exon 10, which includes theNGF binding site, the latter of which is associated with acute myeloidleukemia.

In some examples, a dysregulation of a NTRK gene, a Trk protein, orexpression or activity, or level of the same, can include an alternatespliced form of a Trk mRNA, e.g., a TrkAIII spliced variant or analternative spliced form of a TrkA mRNA that results in the productionof a TrkA protein that lacks the amino acids encoded by exon 10. In someexamples, a dysregulation of a NTRK gene, a Trk protein, or expressionor activity, or level of the same, includes an amplification of a NTRKgene (e.g., one, two, three, or four additional copies of the NTRK gene)that can result, e.g., in an autocrine expression of a NTRK gene in acell.

The term “Trk-associated cancer or tumor” is a cancer that is associatedwith dysregulation of a NTRK gene, a Trk protein, or expression oractivity, or level of the same (e.g., a cancer that is associated withat least one example (e.g., two, three, four, or five examples) ofdysregulation of a NTRK gene, a Trk protein, or expression or activity,or level of the same, described herein).

The term “mammal” as used herein, refers to a warm-blooded animal thathas or is at risk of developing a disease described herein and includes,but is not limited to, guinea pigs, dogs, cats, rats, mice, hamsters,and primates, including humans.

The term “therapeutically effective amount” as used herein, means thatamount of active compound or pharmaceutical agent that elicits thebiological or medicinal response in a tissue system, animal or humanthat is being sought by a researcher, veterinarian, medical doctor orother clinician, which includes alleviation of the symptoms of thedisease or disorder being treated. In particular, a therapeuticallyeffective amount, when administered to a subject in need of suchtreatment, is sufficient to (i) treat or prevent a particular disease,condition, or disorder which can be treated with an inhibitor of TrkAand/or TrkB, (ii) attenuate, ameliorate, or eliminate one or moresymptoms of the particular disease, condition, or disorder, or (iii)prevent or delay the onset of one or more symptoms of the particulardisease, condition, or disorder described herein. The amount ofcrystalline form (I-HS) that will correspond to such a therapeuticallyeffective amount will vary depending upon factors such the diseasecondition and its severity, the identity (e.g., weight) of the mammal inneed of treatment, but can nevertheless be routinely determined by oneskilled in the art.

As used herein, the term “composition” is intended to encompass aproduct comprising the specified ingredients in the specified amounts,as well as any product which results, directly or indirectly, fromcombinations of the specified ingredients in the specified amounts.

To provide a more concise description, some of the quantitativeexpressions given herein are not qualified with the term “about.” It isunderstood that whether the term “about” is used explicitly or not,every quantity given herein is meant to refer to the actual given value,and it is also meant to refer to the approximation to such given valuethat would reasonably be inferred based on the ordinary skill in theart, including approximations due to the experimental and/or measurementconditions for such given value.

In some embodiments, the term “about” is used herein to meanapproximately, in the region of, roughly, or around. When the term“about” is used in conjunction with a numerical range, it modifies thatrange by extending the boundaries above and below the numerical valuesset forth. In general, the term “about” is used herein to modify anumerical value above and below the stated value by a variance of 10%.

The term “about” preceding one or more peak positions in an X-ray powderdiffraction pattern means that all of the peaks of the group which itprecedes are reported in terms of angular positions (two theta) with anallowable variability of ±0.3°. The variability of ±0.3° is intended tobe used when comparing two powder X-ray diffraction patterns. Inpractice, if a diffraction pattern peak from one pattern is assigned arange of angular positions (two theta) which is the measured peakposition ±0.3° and if those ranges of peak positions overlap, then thetwo peaks are considered to have the same angular position. For example,if a peak from one pattern is determined to have a position of 11.0°,for comparison purposes the allowable variability allows the peak to beassigned a position in the range of 10.7°-11.3°.

The term “about” preceding a value for DSC, TGA, TG, or DTA, which arereported as degrees Celsius, have an allowable variability of ±5° C.

To provide a more concise description, some of the quantitativeexpressions herein are recited as a range from about amount X to aboutamount Y. It is understood that wherein a range is recited, the range isnot limited to the recited upper and lower bounds, but rather includesthe full range from about amount X through about amount Y, or any rangetherein.

Further provided herein are pharmaceutical compositions containingcrystalline form (I-HS) with a pharmaceutically acceptable carrier.Pharmaceutical compositions containing crystalline form (I-HS) as theactive ingredient can be prepared by intimately mixing crystalline form(I-HS) with a pharmaceutical carrier according to conventionalpharmaceutical compounding techniques. The carrier may take a widevariety of forms depending upon the desired route of administration(e.g., oral, parenteral). Thus for liquid oral preparations such assuspensions, elixirs and solutions, suitable carriers and additivesinclude water, glycols, oils, alcohols, flavoring agents, preservatives,stabilizers, coloring agents and the like; for solid oral preparations,such as powders, capsules and tablets, suitable carriers and additivesinclude starches, sugars, diluents, granulating agents, lubricants,binders, disintegrating agents and the like. Solid oral preparations mayalso be coated with substances such as sugars or be enteric-coated so asto modulate major site of absorption. For parenteral administration, thecarrier will usually consist of sterile water and other ingredients maybe added to increase solubility or preservation. Injectable suspensionsor solutions may also be prepared utilizing aqueous carriers along withappropriate additives.

Crystalline form (I-HS) may be administered by any convenient route,e.g. into the gastrointestinal tract (e.g. rectally or orally), thenose, lungs, musculature or vasculature, or transdermally or dermally.Crystalline form (I-HS) may be administered in any convenientadministrative form, e.g. tablets, powders, capsules, solutions,dispersions, suspensions, syrups, sprays, suppositories, gels,emulsions, patches etc. Such compositions may contain componentsconventional in pharmaceutical preparations, e.g. diluents, carriers, pHmodifiers, sweeteners, bulking agents, and further active agents. Ifparenteral administration is desired, the compositions will be sterileand in a solution or suspension form suitable for injection or infusion.Such compositions form a further aspect of the invention.

Also provided herein are pharmaceutical compositions comprisingcrystalline form (I-HS). To prepare the pharmaceutical compositionsprovided herein, crystalline form (I-HS) as the active ingredient isintimately admixed with a pharmaceutical carrier according toconventional pharmaceutical compounding techniques, which carrier maytake a wide variety of forms depending of the form of preparationdesired for administration, e.g., oral or parenteral such asintramuscular. In preparing the compositions in oral dosage form, any ofthe usual pharmaceutical media may be employed. Thus, for liquid oralpreparations, such as for example, suspensions, elixirs and solutions,suitable carriers and additives include water, glycols, glycerols, oils,cyclodextrins, alcohols, e.g., ethanol, flavoring agents, preservatives,coloring agents and the like; for solid oral preparations such as, forexample, powders, capsules, caplets, gelcaps and tablets, suitablecarriers and additives include starches, sugars, diluents, granulatingagents, lubricants, binders, disintegrating agents and the like.Suitable binders include, without limitation, starch, gelatin, naturalsugars such as glucose or beta-lactose, corn sweeteners, natural andsynthetic gums such as acacia, tragacanth or sodium oleate, sodiumstearate, magnesium stearate. sodium benzoate, sodium acetate, sodiumchloride and the like. Disintegrators include, without limitation,starch, methyl cellulose, agar, bentonite, xanthan gum and the like.

Because of their ease in administration, tablets and capsules representthe most advantageous oral dosage unit form, in which case solidpharmaceutical carriers are obviously employed. If desired, tablets maybe sugar coated or enteric coated by standard techniques. Forparenterals, the carrier will usually comprise sterile water, throughother ingredients, for example, for purposes such as aiding solubilityor for preservation, may be included. Injectable suspensions may also beprepared, in which case appropriate liquid carriers, suspending agentsand the like may be employed. The pharmaceutical compositions hereinwill contain, per dosage unit, e.g., tablet, capsule, powder, injection,teaspoonful and the like, an amount of the active ingredient necessaryto deliver an effective dose as described above.

The pharmaceutical compositions herein will contain, per unit dosageunit, e.g., tablet, capsule, suspension, solution, sachet forreconstitution, powder, injection, I.V., suppository, sublingual/buccalfilm, teaspoonful and the like, of from about 0.1-1000 mg or any rangetherein, and may be given at a dosage of from about 0.01-300 mg/kg/day,or any range therein, preferably from about 0.5-50 mg/kg/day, or anyrange therein. In some embodiments, the pharmaceutical compositionsprovided herein contain, per unit dosage unit, about 25 mg to about 500mg of a compound provided herein (for example, about 25 mg to about 400mg, about 25 mg to about 300 mg, about 25 mg to about 250 mg, about 25mg to about 200 mg, about 25 mg to about 150 mg, about 25 mg to about100 mg, about 25 mg to about 75 mg, about 50 mg to about 500 mg, about100 mg to about 500 mg, about 150 mg to about 500 mg, about 200 mg toabout 500 mg, about 250 mg to about 500 mg, about 300 mg to about 500mg, about 400 mg to about 500 mg, about 50 to about 200 mg, about 100 toabout 250 mg, about 50 to about 150 mg). In some embodiments, thepharmaceutical compositions provided herein contain, per unit dosageunit, about 25 mg, about 50 mg, about 100 mg, about 150 mg, about 200mg, about 250 mg, about 300 mg, about 400 mg, or about 500 mg of acompound provided herein. The dosages, however, may be varied dependingupon the requirement of the patients, the severity of the conditionbeing treated and the compound being employed. In some embodiments, thedosages are administered once daily (QD) or twice daily (BID).

Preferably these compositions are in unit dosage forms from such astablets, pills, capsules, powders, granules, sterile parenteralsolutions or suspensions, metered aerosol or liquid sprays, drops,ampoules, autoinjector devices or suppositories; for oral parenteral,intranasal, sublingual or rectal administration, or for administrationby inhalation or insufflation. Alternatively, the composition may bepresented in a form suitable for once-weekly or once-monthlyadministration; for example, an insoluble salt of the active compound,such as the decanoate salt, may be adapted to provide a depotpreparation for intramuscular injection. For preparing solidcompositions such as tablets, crystalline form (I-HS) is mixed with apharmaceutical carrier, e.g. conventional tableting ingredients such ascorn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesiumstearate, dicalcium phosphate or gums, and other pharmaceuticaldiluents, e.g. water, to form a solid preformulation compositioncontaining a homogeneous mixture of crystalline form (I-HS). Whenreferring to these preformulation compositions as homogeneous, it ismeant that the active ingredient is dispersed evenly throughout thecomposition so that the composition may be readily subdivided intoequally effective dosage forms such as tablets, pills and capsules. Thissolid preformulation composition is then subdivided into unit dosageforms of the type described above containing from 0.1 to about 1000 mg,or any amount or range thereof, of the active ingredient providedherein. The tablets or pills of the novel composition can be coated orotherwise compounded to provide a dosage form affording the advantage ofprolonged action. For example, the tablet or pill can comprise an innerdosage and an outer dosage component, the latter being in the form of anenvelope over the former. The two components can be separated by anenteric layer which serves to resist disintegration in the stomach andpermits the inner component to pass intact into the duodenum or to bedelayed in release. A variety of material can be used for such entericlayers or coatings, such materials including a number of polymeric acidswith such materials as shellac, cetyl alcohol and cellulose acetate.

The liquid forms in which the novel compositions provided herein may beincorporated for administration orally or by injection include, aqueoussolutions, cyclodextrins, suitably flavored syrups, aqueous or oilsuspensions, and flavored emulsions with edible oils such as cottonseedoil, sesame oil, coconut oil or peanut oil, as well as elixirs andsimilar pharmaceutical vehicles. Suitable dispersing or suspendingagents for aqueous suspensions, include synthetic and natural gums suchas tragacanth, acacia, alginate, dextran, sodium carboxymethylcellulose,methylcellulose, polyvinyl-pyrrolidone or gelatin. For parenteraladministration, sterile suspensions and solutions are desired. Isotonicpreparations which generally contain suitable preservatives are employedwhen intravenous administration is desired.

Crystalline form (I-HS) can be administered in intranasal form viatopical use of suitable intranasal vehicles, or via transdermai skinpatches well known to those of ordinary skill in that art. To beadministered in the form of a transdermal delivery system, the dosageadministration will, of course, be continuous rather than intermittentthroughout the dosage regimen.

To prepare a pharmaceutical compositions provided herein, crystallineform (I-HS) as the active ingredient is intimately admixed with a.pharmaceutical carrier according to conventional pharmaceuticalcompounding techniques, which carrier may take a wide variety of formsdepending of the form of preparation desired for administration e.g.oral or parenteral). Suitable pharmaceutically acceptable carriers arewell known in the art. Descriptions of some of these pharmaceuticallyacceptable carriers may be found in The Handbook of PharmaceuticalExcipients, published by the American Pharmaceutical Association and thePharmaceutical Society of Great Britain.

Methods of formulating pharmaceutical compositions have been describedin numerous publications such as Pharmaceutical Dosage Forms: Tablets,Second Edition Revised and Expanded, Volumes 1-3, edited by Lieberman etal; Pharmaceutical Dosage Forms: Parenteral Medications, Volumes 1-2,edited by Avis et al; and Pharmaceutical Dosage Forms: Disperse Systems,Volumes 1-2, edited by Lieberman et al; published by Marcel Dekker, Inc.

Compounds provided herein may be administered in any of the foregoingcompositions and according to dosage regimens established in the artwhenever treatment of cancer, pain, inflammation, neurodegenerativedisease or Trypanosoma cruzi infection s required.

The daily dosage of crystalline form (I-HS) may be varied over a widerange from 1.0 to 10,000 mg per adult human per day, or higher, or anyrange therein. For oral administration, the compositions are preferablyprovided in the form of tablets containing, 0.01, 0.05, 0.1, 0.5, 1.0,2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 150, 200, 250 and 500 milligramsof the active ingredient for the symptomatic adjustment of the dosage tothe patient to be treated. An effective amount of the drug is ordinarilysupplied at a dosage level of from about 0.1 mg/kg to about 1000 mg/kgof body weight per day, or any range therein. Preferably, the range isfrom about 0.5 to about 500 mg/kg of body weight per day, or any rangetherein. More preferably, from about 1.0 to about 250 mg/kg of bodyweight per day, or any range therein. More preferably, from about 0.1 toabout 100 mg/kg of body weight per day, or any range therein. In anexample, the range may be from about 0.1 to about 50.0 mg/kg of bodyweight per day, or any amount or range therein. In another example, therange may be from about 0.1 to about 15.0 mg/kg of body weight per day,or any range therein. In yet another example, the range may be fromabout 0.5 to about 7.5 mg/kg of body weight per day, or any amount torange therein. Crystalline form (I-HS) may be administered on a regimenof 1 to 4 times per day or in a single daily dose.

Optimal dosages to be administered may be readily determined by thoseskilled in the art, and will vary with the mode of administration, thestrength of the preparation, the mode of administration, and theadvancement of the disease condition. In addition, factors associatedwith the particular patient being treated, including patient age,weight, diet and time of administration, will result in the need toadjust dosages.

One skilled in the art will recognize that, both in vivo and in vitrotrials using suitable, known and generally accepted cell and/or animalmodels are predictive of the ability of a test compound to treat orprevent a given disorder.

One skilled in the art will further recognize that human clinical trialsincluding first-in-human, dose ranging and efficacy trials, in healthypatients and/or those suffering from a given disorder, may be completedaccording to methods well known in the clinical and medical arts.

Acronyms found in the specification have the following meanings:

ATP adenosine triphosphate DI deionized EtOH ethanol GC gaschromatography MOPS 3-(N-morpholino)-propanesulfonic acid MTBE methyltert-butyl ether PDA photodiode array RRT relative retention time RTroom temperature THF tetrahydrofuran TMB 3,3′,5,5′-tetramethylbenzidine

The following examples illustrate the invention and are set forth to aidin the understanding of the invention, and are not intended and shouldnot be construed to limit in any way the invention set forth in theclaims which follow thereafter.

In the examples described below, unless otherwise indicated alltemperatures are set forth in degrees Celsius. Reagents were purchasedfrom commercial suppliers such as Sigma-Aldrich Chemical Company, EMD,JT Baker, or Pharco-Aaper, and were used without further purificationunless otherwise indicated. Tetrahydrofuran (THF), heptane and otherorganic solvents were purchased from commercial suppliers, such asSigma-Aldrich Chemical Company, ACROS, Alfa-Aesar, Lancaster, TCI, orMaybridge, and used as received.

One skilled in the art will recognize that, where not otherwisespecified, the reaction step(s) is performed under suitable conditions,according to known methods, to provide the desired product. One skilledin the art will also recognize that wherein a reaction step as disclosedherein may be carried out in a variety of solvents or solvent systems,said reaction step may also be carried out in a mixture of the suitablesolvents or solvent systems. One skilled in the art will recognize that,in the specification and claims as presented herein, wherein a reagentor reagent class/type (e.g. base, solvent, etc.) is recited in more thanone step of a process, the individual reagents are independentlyselected for each reaction step and may be the same of different fromeach other. For example wherein two steps of a process recite an organicor inorganic base as a reagent, the organic or inorganic base selectedfor the first step may be the same or different than the organic orinorganic base of the second step.

The reactions set forth below were done generally under a positivepressure of nitrogen (unless otherwise stated) in “ACS grade” solvents,and the reaction flasks were typically fitted with rubber septa for theintroduction of substrates and reagents via syringe or addition funnel.

Two reversed-phase high performance liquid chromatography (HPLC) systemswere used for in-process monitoring and analysis, using acetonitrile andwater/trifluoroacetic acid as mobile phases. One system employed anAgilent Zorbax Extend C18 column at 264 nm, while the other system(hereinafter, “TRK1PM1 HPLC”) included a Waters Xbridge Phenyl Column at268 nm. Unless otherwise specified, the former system was used. Thesilica for both systems was stirred in a flask with the compound, andthen filtered through a polypropylene cloth before being analyzed.

Amorphous freebase form of compound of Formula I: About 1 gram of(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamideis dissolved in minimum amount of water and cooled to a temperature ofabout −26° Celsius followed by drying in the freeze dryer for 24 hours.About 20 mg of the amorphous material obtained from the freeze dryer wasweighed in a vial, to which 5 volume aliquots of an appropriate solventsystem was added. The mixture was checked for dissolution and if nodissolution was apparent, the mixture was heated to about 40° Celsiusand checked again. This procedure was continued until dissolution wasobserved or until 100 volumes of solvent had been added. The XRPDpattern of the amorphous material obtained from the freeze dryingexperiment is shown in FIG. 7.

Amorphous hydrogen sulfate salt of compound of Formula I was prepared asdescribed in Example 14A in WO 2010/048314 (see Example 3). The XRPDpatterns of the two different lots of amorphous material prepared bythis method are show in FIG. 28.

Also provided herein is a process for the preparation of crystallineform (I-HS). In some embodiments, the process comprises the steps asshown in Scheme 1.

In some embodiments, provided herein is a process for the preparation ofcrystalline form (I-HS), comprising:

(a) adding concentrated sulfuric acid to a solution of(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidein EtOH to form the hydrogen sulfate salt of(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide;

(b) adding heptane to the solution in Step (a) to form a slurry;

(c) filtering the slurry to isolate(S)—N-(5((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehydrogen sulfate;

(d) mixing said(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehydrogen sulfate with a 5:95 w/w solution of water/2-butanone;

(e) heating the mixture from step (d) at about 65-70° C. with stirringuntil the weight percent of ethanol is about 0.5% to form a slurry ofthe crystalline form of(S)—N-(5-((R-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehydrogen sulfate; and

(f) isolating the crystalline form of(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehydrogen sulfate by filtration.

In some embodiments, the above method further comprises: (b1) seedingthe solution from step (a) with(S)—N-(5((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehydrogen sulfate at room temperature and allowing the solution to stiruntil a slurry forms.

In some embodiments, provided herein is a process for the preparation ofcrystalline form (I-HS), comprising:

(a) reacting 5-chloro-3-nitropyrazolo[1,5-a]pyrimidine with(R)-2-(2,5-difluorophenyl)-pyrrolidin (R)2-hydroxysuccinate in thepresence of a base to form(R)-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)-3-nitropyrazolo[1,5-a]pyrimidine;

(b) treating said(R)-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)-3-nitropyrazolo[1,5-a]pyrimidinewith Zn and hydrochloric acid to form(R)-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-amine;

(c) treating said(R)-5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-aminewith a base and phenyl chloroformate to form phenyl(R)-(5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)carbamate;

(d) reacting said phenyl(R)-(5-(2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)carbamatewith (S)-pyrrolidin-3-ol to form(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-boxamide;

(e) adding sulfuric acid to said(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamideform(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehydrogen sulfate; and

(f) isolating the crystalline form of(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehydrogen sulfate.

In some embodiments of the above step (a), the base is an amine base,such as triethylamine.

In some embodiments of the above step (c), the base is an alkali metalbase, such as an alkali metal carbonate, such as potassium carbonate.

Preparation A

Preparation of 5-chloro-3-nitropyrazolo[1,5-a]pyrimidine

Step A—Preparation of sodium pyrazolo[1,5-alpyrimidin-5-olate: Asolution of 1H-pyrazol-5-amine and1,3-dimethylpyrimidine-2,4(1H,3H)-dione (1.05 equiv.) were charged to around bottom flask outfitted with a mechanical stirrer, a steam pot, areflux condenser, a J-Kem temperature probe and an N₂ adaptor forpositive N₂ pressure control. Under mechanical stirring the solids weresuspended with 4 vol. (4 mL/g) of absolute EtOH under a nitrogenatmosphere, then charged with 2.1 equivalents of NaOEt (21 wt % solutionin EtOH), and followed by line-rinse with 1 vol. (1 mL/g) of absoluteEtOH. The slurry was warmed to about 75° Celsius and stirred at gentlereflux until less than 1.5 area % of 1H-pyrazol-5-amine was observed byTRK1PM1 HPLC to follow the progression of the reaction using 20 μL ofslurry diluted in 4 mL deionized water and 5 μL injection at 220 nm.

After 1 additional hour, the mixture was charged with 2.5 vol. (2.5mL/g) of heptane and then refluxed at 70° Celsius for 1 hour. The slurrywas then cooled to room temperature overnight. The solid was collectedby filtration on a tabletop funnel and polypropylene filter cloth. Thereactor was rinsed and charged atop the filter cake with 4 vol. (4 mL/g)of heptane with the cake pulled and the solids being transferred totared drying trays and oven-dried at 45° Celsius under high vacuum untiltheir weight was constant. Pale yellow solid sodiumpyrazolo[1,5-a]-pyrimidin-5-olate was obtained in 93-96% yield(corrected) and larger than 99.5 area % observed by HPLC (1 mg/mLdilution in deionized water, TRK1PM1 at 220 nm).

Step B—Preparation of 3-nitropyrazolo[1,5-alpyrimidin-5(4H)-one: A taredround bottom flask was charged with sodiumpyrazolo[1,5-a]pyrimidin-5-olate that was dissolved at 40-45° Celsius in3.0 vol. (3.0 mL/g) of deionized water, and then concentrated under highvacuum at 65° Celsius in a water-bath on a rotary evaporator until 2.4×weight of starting material was observed (1.4 vol/1.4 mL/g deionizedwater content). Gas chromatography (GC) for residual EtOH (30 μL ofsolution dissolved in ˜1 mL MeOH) was performed showing less than 100ppm with traces of ethyl nitrate fumes being observed below upon lateraddition of HNO₃. In some cases, the original solution was charged withan additional 1.5 vol. (1.5 mL/g) of DI water, then concentrated underhigh vacuum at 65° Celsius in a water-bath on a rotary evaporator until2.4× weight of starting material was observed (1.4 vol/1.4 mL/g DI watercontent). Gas chromatograph for residual EtOH (30 μL of solutiondissolved in about 1 mL MeOH) was performed showing <<100 ppm ofresidual EtOH without observing any ethyl nitrate fumes below upon lateraddition of HNO₃.

A round bottom vessel outfitted with a mechanical stirrer, a steam pot,a reflux condenser, a J-Kem temperature probe and an N₂ adaptor forpositive N₂ pressure control was charged with 3 vol. (3 mL/g, 10 equiv)of >90 wt % HNO₃ and cooled to about 10° Celsius under a nitrogenatmosphere using external ice-water cooling bath under a nitrogenatmosphere. Using a pressure equalizing addition funnel, the HNO₃solution was charged with the 1.75-1.95 volumes of a deionized watersolution of sodium pyrazolo[1,5-a]pyrimidin-5-olate (1.16-1.4 mL DIwater/g of sodium pyrazolo[1,5-a]pyrimidin-5-olate) at a rate tomaintain 35-40° Celsius internal temperature under cooling. Twoazeotropes were observed without any ethyl nitrate fumes. The azeotropeflask, the transfer line (if applicable) and the addition funnel wererinsed with 2×0.1 vol. (2×0.1 mL/g) deionized water added to thereaction mixture. Once the addition was complete, the temperature wasgradually increased to about 45-50° Celsius for about 3 hours with HPLCshowing >99.5 area % conversion of sodiumpyrazolo[1,5-a]pyrimidin-5-olate to3-nitropyrazolo[1,5-a]pyrimidin-5(4H)-one.

Step C—Preparation of 5-chloro-3-nitropyrazolo[1,5-alpyrimidine:3-nitropyrazolo]1,5-a]pyrimidin-5(4H)-one was charged to a round bottomflask outfitted with a mechanical stirrer, a heating mantle, a refluxcondenser, a J-Kem temperature probe and an N₂ adaptor for positive N₂pressure control. Under mechanical stirring the solids were suspendedwith 8 volumes (8 mL/g) of CH₃CN, and then charged with 2,6-lutitine(1.05 equiv) followed by warming the slurry to about 50° Celsius. Usinga pressure equalizing addition funnel, the mixture was dropwise chargedwith 0.33 equivalents of POCl. This charge yielded a thick, beige slurryof a trimer that was homogenized while stirring until a semi-mobile masswas observed. An additional 1.67 equivalents of POCl was charged to themixture while allowing the temperature to stabilize, followed by warmingthe reaction mixture to a gentle reflux (78° Celsius). Some puffing wasobserved upon warming the mixture that later subsided as the thickslurry got thinner.

The reaction mixture was allowed to reflux until complete dissolution toa dark solution and until HPLC (20 μL diluted in 5 mL of CH₃CN, TRK1PM1HPLC, 5 μL injection, 268 nm) confirmed that no more trimer (RRT 0.92)was present with less than 0.5 area % of3-nitropyrazolo[1,5-a]pyrimidin-5(4H)-one (RRT 0.79) being observed bymanually removing any interfering and early eluting peaks related tolutidine from the area integration. On a 1.9 kg scale, 0 area % of thetrimer, 0.25 area % of 3-nitropyrazolo[1,5-a]pyrimidin-5(4H)-one, and99.5 area % of 5-chloro-3-nitropyrazolo[1,5-a]pyrimidine was observedafter 19 hours of gentle reflux using TRK1PM1 HPLC at 268 nm

Preparation B

Preparation of (R)-2-(2,5-difluorophenyl)-pyrrolidine(R)-2-hydroxysuccinate

Step A—Preparation of tert-butyl(4-(2,5-difluorophenyl)-4-oxobutyl)-carbamate:2-bromo-1,4-difluorobenzene (1.5 eq.) was dissolved in 4 volumes of THF(based on weight of tert-butyl 2-oxopyrrolidine-1-carboxylate) andcooled to about 5° Celsius. A solution of 2.0 M iPrMgCl in THF (1.4 eq.)was added over 2 hours to the mixture while maintaining a reactiontemperature below 25° Celsius. The solution was allowed to cool to about5° Celsius and stirred for 1 hour (GC analysis confirmed Grignardformation). A solution of tert-butyl 2-oxopyrrolidine-1-carboxylate (1.0eq.) in 1 volume of THF was added over about 30 min while maintaining areaction temperature below 25° Celsius. The reaction was stirred atabout 5° Celsius for 90 min (tert-butyl 2-oxopyrrolidine-1-carboxylatewas confirmed to be less than 0.5 area % by HPLC). The reaction wasquenched with 5 volumes of 2 M aqueous HCl while maintaining a reactiontemperature below 45° Celsius. The reaction was then transferred to areparatory funnel adding 10 volumes of heptane and removing the aqueouslayer. The organic layer was washed with 4 volumes of saturated aqueousNaCl followed by addition of 2×1 volume of saturated aqueous NaCl. Theorganic layer was solvent-switched to heptane (<1% wt THF confirmed byGC) at a distillation temperature of 35-55° Celsius and distillationpressure of 100-200 mm Hg for 2×4 volumes of heptane being added with aminimum distillation volume of about 7 volumes. The mixture was thendiluted to 10 volumes with heptane while heating to about 55° Celsiusyielded a denser solid with the mixture being allowed to cool to roomtemperature overnight. The slurry was cooled to less than 5° Celsius andfiltered through polypropylene filter cloth. The wet cake was washedwith 2×2 volumes of heptane. The solids were dried under vacuum at 55°Celsius until the weight was constant, yielding tent-butyl(4-(2,5-difluorophenyl)-4-oxobutyl)-carbamate as a white solid at about75% to 85% theoretical yield.

Step B—Preparation of 5-(2,5-difluorophenyl)-3,4-dihydro-2H-pyrrole:tent-butyl (4-(2,5-difluorophenyl)-4-oxobutyl)-carbamate was dissolvedin 5 vol. of toluene with 2.2 eq. of 12M HCl being added observing amild exotherm and gas evolution. The reaction was heated to 65° Celsiusfor 12-24 hours and monitored by HPLC. Upon completion the reaction wascooled to less than 15° Celsius with an ice/water bath. The pH wasadjusted to about 14 with 3 equivalents of 2M aqueous NaOH (4.7 vol.).The reaction was stirred at room temperature for 1-2 hours. The mixturewas transferred to a separatory funnel with toluene. The aqueous layerwas removed and the organic layer was washed with 3 volumes of saturatedaqueous NaCl. The organic layer was concentrated to an oil andredissolved in 1.5 volumes of heptane. The resulting suspension wasfiltered through a GF/F filter paper and concentrated to a light yellowoil of 5-(2,5-difluorophenyl)-3,4-dihydro-2H-pyrrole with a 90% to 100%theoretical yield.

Step C—Preparation of (R)-2-(2,5-difluorophenyl)-pyrrolidine:Chloro-1,5-cyclooctadiene iridium dimer (0.2 mol %) and(R)-2-(2-(diphenylphosphino)phenyl)-4-isopropyl-4,5-dihydrooxazole (0.4mol %) were suspended in 5 volumes of MTBE (based on5-(2,5-difluorophenyl)-3,4-dihydro-2H-pyrrole) at room temperature. Themixture was stirred for 1 hour and most of the solids dissolved with thesolution turning dark red. The catalyst formation was monitored using anHPLC/PDA detector. The reaction was cooled to less than 5° Celsius and5-(2,5-difluorophenyl)-3,4-dihydro-2H-pyrrole (1.0 eq.) was added usinga 0.5 volumes of MTBE rinse. Diphenylsilane (1.5 eq.) was added overabout 20 minutes while maintaining a reaction temperature below 10°Celsius. The reaction was stirred for 30 minutes below 10° Celsius andthen allowed to warm to room temperature. The reaction was stirredovernight at room temperature. The completion of the reaction wasconfirmed by HPLC and then cooled to less than 5° Celsius. The reactionwas quenched with 5 volumes of 2M aqueous HCl maintaining temperaturebelow 20° Celsius. After 10 minutes the ice/water bath was removed andthe reaction temperature was allowed to increase to room temperaturewhile stirring for 2 hours. The mixture was transferred to a separatoryfunnel with 3 volumes of MTBE. The aqueous layer was washed with 3.5volumes of MTBE followed by addition of 5 volumes of MTBE to the aqueouslayer while adjusting the pH to about 14 by adding 0.75 volumes ofaqueous 50% NaOH. The organic layer was washed with 5 volumes of aqueoussaturated NaCl, then concentrated to an oil, and diluted with 3 volumesof MTBE. The solution was filtered through a polypropylene filter clothand rinsed with 1 volume of MTBE. The filtrate was concentrated to anoil of (R)-2-(2,5-difluorophenyl)-pyrrolidine with a 95% to 100%theoretical yield and with 75-85% ee.

Step D—Preparation of (R)-2-(2,5-difluorophenyl)-pyrrolidine(R)-2-hydroxy-succinate: (R)-2-(2,5-difluorophenyl)-pyrrolidine (1.0eq.) was transferred to a round bottom flask charged with 15 volumes(corrected for potency) of EtOH (200 prf). D-malic acid (1.05 eq.) wasadded and the mixture was heated to 65° Celsius. The solids alldissolved at about 64° Celsius. The solution was allowed to cool to RT.At about 55° Celsius the solution was seeded with(R)-2-(2,5-difluorophenyl)-pyrrolidine (R)-2-hydroxy-succinate (about 50mg, >97% ee) and stirred at room temperature overnight. The suspensionwas then filtered through a polypropylene filter cloth and washed with2×1 volumes of EtOH (200 prf). The solids were dried under vacuum at 55°Celsius, yielding (R)-2-(2,5-difluorophenyl)-pyrrolidine(R)-2-hydroxy-succinate with a 75% to 90% theoretical yield andwith >96% ee.

Referring to Scheme 1, suitable bases include tertiary amine bases, suchas triethylamine, and K₂CO₃. Suitable solvents include ethanol, heptaneand tetrahydrofuran (THF). The reaction is conveniently performed attemperatures between 5° Celsius and 50° Celsius. The reaction progresswas generally monitored by HPLC TRK1PM1.

Compounds II (5-chloro-3-nitropyrazolo[1,5-a]pyrimidine) and III((R)-2-(2,5-difluorophenyl)pyrrolidine (R)-2-hydroxysuccinate, 1.05 eq.)were charged to a round bottom flask outfitted with a mechanicalstirrer, a J-Kem temperature probe and an N₂ adaptor for positive N₂pressure control. A solution of 4:1 EtOH:THF (10 mL/g of compound II)was added and followed by addition of triethylamine (NEt₃, 3.50 eq.) viaaddition funnel with the temperature reaching about 40° Celsius duringaddition. Once the addition was complete, the reaction mixture washeated to 50° Celsius and stirred for 0.5-3 hours to yield compound IV.

To a round bottom flask equipped with a mechanical stirrer, a J-Kemtemperature probe, and an N₂ inlet compound IV was added and followed byaddition of tetrahydrofuran (10 mL/g of compound IV). The solution wascooled to less than 5° Celsius in an ice bath, and Zn (9-10 eq.) wasadded. 6M HCl (9-10 eq.) was then added dropwise at such a rate to keepthe temperature below 30° Celsius (for 1 kg scale the addition tookabout 1.5 hours). Once the exotherm subsided, the reaction was allowedto warm to room temperature and was stirred for 30-60 min until compoundIV was not detected by HPLC. At this time, a solution of potassiumcarbonate (K₂CO₃, 2.0 eq.) in water (5 mL/g of compound IV) was addedall at once and followed by rapid dropwise addition of phenylchloroformate (PhOCOCl, 1.2 eq.). Gas evolution (CO₂) was observedduring both of the above additions, and the temperature increased toabout 30° Celsius after adding phenyl chloroformate. The carbamateformation was stirred at room temperature for 30-90 min. HPLC analysisimmediately followed to run to ensure less than 1 area % for the aminebeing present and high yield of compound VI in the solution.

To the above solution amine VII ((S)-pyrrolidin-3-ol, 1.1 eq. based ontheoretical yield for compound VI) and EtOH (10 mL/g of compound VI) wasadded. Compound VII was added before or at the same time as EtOH toavoid ethyl carbamate impurities from forming. The above EtOH solutionwas concentrated to a minimum volume (4-5 mL/g) using the batchconcentrator under reduced pressure (THF levels should be <5% by GC),and EtOH (10 mL/g of compound VI) was back-added to give a total of 10mL/g. The reaction was then heated at 50° Celsius for 9-19 hours oruntil HPLC shows that compound VI is less than 0.5 area %. The reactionwas then cooled to room temperature, and sulfuric acid (H₂SO₄, 1.0 eq.to compound VI) was added via addition funnel to yield compound I-HSwith the temperature usually exotherming at about 30° Celsius.

EXAMPLE 1 Preparation of Crystalline Form (I-HS) (Method 1)

(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide(0.500 g, 1.17 mmol) was dissolved in EtOH (2.5 mL) and cooled to about5° Celsius. Concentrated sulfuric acid (0.0636 mL, 1.17 mmol) was addedto the cooled solution and stirred for about 10 min, while warming toroom temperature. Methyl tert-butyl ether (MTBE) (2 mL) was slowly addedto the mixture, resulting in the product gumming out. EtOH (2.5 mL) wasthen added to the mixture and heated to about reflux until all solidswere dissolved. Upon cooling to room temperature and stirring for about1 hour, some solids formed. After cooling to about 5° Celsius, thesolids were filtered and washed with MTBE. After filtration and dryingat air for about 15 minutes,(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehydrogen sulfate was isolated as a solid.

EXAMPLE 2 Preparation of Crystalline Form (I-HS) (Method 2)

Concentrated sulfuric acid (392 mL) was added to a solution of 3031 g of(S)—N-(5-((R-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidein 18322 mL EtOH to form the hydrogen sulfate salt. The solution wasseeded with 2 g of(S)—N-(5((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehydrogen sulfate and the solution was stirred at room temperature for atleast 2 hours to form a slurry of the hydrogen sulfate salt. Heptane(20888 g) was added and the slurry was stirred at room temperature forat least 60 min. The slurry was filtered and the filter cake was washedwith 1:1 heptane/EtOH. The solids were then dried under vacuum atambient temperature (oven temperature set at 15° Celsius).

The dried hydrogen sulfate salt (6389 g from 4 combined lots) was addedto a 5:95 w/w solution of water/2-butanone (total weight 41652 g). Themixture was heated at about 68° Celsius with stirring until the weightpercent of ethanol was about 0.5%, during which time a slurry formed.The slurry was filtered, and the filter cake was washed with a 5:95 w/wsolution of water/2-butanone. The solids were then dried under vacuum atambient temperature (oven temperature set at 15° Celsius) to provide thecrystalline form of(S)—N-(5-((R)-2-(2,5-difluorophenyl)-pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehydrogen sulfate.

EXAMPLE 3 Preparation of Amorphous Form AM(HS)

To a solution of(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide(9.40 g, 21.94 mmol) in MeOH (220 mL) was slowly added sulfuric acid(0.1 M in MeOH, 219.4 mL, 21.94 mmol) at ambient temperature under rapidstirring. After 30 minutes, the reaction was first concentrated byrotary evaporator to near dryness, then on high vacuum for 48 h toprovide amorphous form of(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidesulfate (11.37 g, 21.59 mmol, 98.43% yield). LCMS (apci m/z 429.1, M+H).

EXAMPLE 4 Preparation of Crystalline HCl Salt of Formula I

A mixture of(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide(0.554 g, 1.29 mmol) in EtOH (6 mL, 200 proof) and MTBE (10 mL) washeated to 50 ° C. while stirring to obtain a solution, followed byaddition of hydrogen chloride (conc.) (0.108 mL, 1.29 mmol) in oneportion. The reaction mixture was then allowed to cool to ambienttemperature first, then cooled to about 5° C. in an ice-water bath withstirring to induce crystallization. The suspension was stirred for 4 hin the ice-water bath before it was vacuum-filtered, with the filtercake rinsed with MTBE and dried under vacuum at 55° C. to constantweight, yielding crystalline(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidine-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide hydrochloride(0.534 g, 89% yield). LCMS (apci m/z 429.2, M+H).

Preparation of Crystalline HBr Salt of Formula I

A mixture of (S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide(0.505 g, 1.18 mmol) in EtOH (6 mL, 200 proof) and MTBE (10 mL) washeated to 50° C. while stirring to obtain a solution, followed byaddition of hydrogen bromide (33% aq.) (0.213 mL, 1.18 mmol) in oneportion. The reaction mixture was heated to reflux to obtain a mostlyclear solution with small amount of oily residue on glass wall ofreaction vessel. Upon cooled to ambient temperature, precipitationappeared and the oily residue solidified. The mixture was heated to 50°C. again, then allowed to cool to room temperature and stirred forovernight. The suspension was vacuum-filtered, with the filter cakerinsed with MTBE and dried under vacuum at 55° C. to constant weight,yielding crystalline(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehydrobromide (0.51 g, 85% yield). LCMS (apci m/z 429.3, M+H).

Preparation of Crystalline Mesylate Salt of Formula I

A mixture of(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide(0.532 g, 1.24 mmol) in EtOH (2.7 mL, 200 proof) and MTBE (5.3 mL) washeated to 50° C. while stirring to obtain a solution, followed byaddition of methanesulfonic acid (0.076 mL, 1.24 mmol) in one portion.The reaction mixture was heated to reflux to obtain a mostly clearsolution with small amount of particulates. Upon cooled to ambienttemperature, precipitation appeared along with some oily residue.Additional EtOH (0.5 mL, 200-proof) and methanesulfonic acid (0.010 mL)were added to obtain a solution. The reaction mixture was heated to 50°C. again, then allowed to cool to room temperature and stirred for 1 h.The suspension was vacuum-filtered, with the filter cake rinsed withMTBE and dried under vacuum at 55° C. to constant weight, yieldingcrystalline(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidemethanesulfonate (0.51 g, 78% yield). LCMS (apci m/z 429.4, M+H).

Preparation of Crystalline Camsylate Salt of Formula I

A mixture of(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide(0.500 g, 1.17 mmol) and S-(+)-camphorsulfonic acid (0.271 g, 1.17 mmol)in EtOH (3 mL, 200 proof) and MTBE (5 mL) was heated to reflux whilestirring to obtain a solution. Upon cooled to ambient temperature,precipitation appeared. The suspension was stirred at room temperaturefor overnight, then vacuum-filtered, with the filter cake rinsed withMTBE and dried under vacuum at 55° C. to constant weight, yieldingcrystalline(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide((1S , 4R)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptan-1-yl)methanesulfonate.

EXAMPLE 5 Comparison of(S)—N-(5((R)-2-(2,5-difluorophenyl)-pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidesalts

Other salt forms of(S)—N-(5((R)-2-(2,5-difluorophenyl)-pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide,e.g., hydrogen chloride, hydrogen bromide, mesylate, and camsylate salts(see Example 4), were compared to crystalline form (I-HS) by determiningtheir differential scanning calorimetry (DSC) melting point, dynamicvapor sorption (DVS) weight gain and stability on an aluminum slide at40° Celsius and 75% relative humidity (RH). The DSC and DVS measurementwere performed as described above with the results being summarized inTable 14.

TABLE 14 Physicochemical Properties of Crystalline Salts of (S)-N-(5-((R)-2-(2,5-difluorophenyl)-pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide DSC MeltingStability at 40° Crystalline Point onset DVS weight gain Celsius/75% RHSalt to max (outcome) (aluminum slide) Hydrogen 186-206° Celsius ~1%gain at 80% 10 days Sulfate RH No form change (I-HS) A 2% gain at 95% RHNo change HCl 124-134° Celsius ~1% gain at 50% After 1 hour a RH formchange at 50-60% RH a occurred form change occurred HBr 177-185° Celsius~3.9% gain at 80% After 2 weeks RH became ~25% gain at 90% amorphous RH(deliquesced) Mesylate 183-186° Celsius ~9% gain at 80% Deliquesced RHovernight ~50% gain at 90% RH (crystalline) Camsylate 170-183° CelsiusNot Tested Not Tested

EXAMPLE 6 TrkA and TrkB Enzyme Assay

The affinity of a compound binding to Trk kinase is measured usingInvitrogen's LanthaScreen™ Eu Kinase Binding technology. Briefly,His-tagged recombinant human Trk cytoplasmic domain from Invitrogen (5nM TRK A—Cat. No. PV3144 or 10 nM TRK B—Cat. No. PV3616) is incubatedwith 5 nM Alexa-Fluor® Tracer 236 (PR9078A), 2 nM biotinylated anti-His(Cat. No. M4408), and 2 nM europium-labeled Streptavidin (Cat No.PV5899) along with test compound in a buffer consisting of 25 mM MOPS,pH 7.5, 5 mM MgCl₂, 0.005% Triton X-100, and 2% DMSO. The compound istypically prepared in a three-fold serial dilution in DMSO and added tothe assay to give the appropriate final concentration. After a 60-minuteincubation at 22° C., the reaction is measured using a PerkinElmerEnVision multimode plate reader via TR-FRET dual wavelength detection,and the percent of control (POC) calculated using a ratiometric emissionfactor. 100 POC is determined using no test compound and 0 POC isdetermined using a concentration of control compound that completelyinhibits the enzyme. The POC values are fit to a 4 parameter logisticcurve and the IC₅₀ value is point where the curve crosses 50 POC.Crystalline form (I-HS) had an averaged IC₅₀ of 8.4 nM when tested inthis assay for TrkA and an averaged IC₅₀ of 4.2 when tested in thisassay for TrkB.

EXAMPLE TRK Fusion Proteins Drive Oncogenesis and are Inhibited by theCrystalline Form (I-HS)

A set of experiments were performed to determine whether the crystallineform (I-HS) would inhibit cell proliferation in three different cancercell line models harboring different Trk gene fusions: CUTO-3F cellline, KM12 cell line, and a MO-91 cell line. The CUTO-3F cell is derivedfrom a patient with lung adenocarcinoma harboring the MPRIP-NTRK1 genefusion. The KM12 cell line is a colorectal cancer cell line harboringthe TPM3-NTRK1 fusion (Vaishnavi et al., Nature Med. 19:1469-1472,2013). The MO-91 cell line is derived from an acute myeloid leukemiapatient harboring the ETV6-NTRK3 fusion (Taipale et al., Nature Biotech.31:630-637, 2013). Measurement of the proliferation of the cellsfollowing treatment with the crystalline form (I-HS) demonstrated adose-dependent inhibition of cell proliferation in all three tested celllines (FIGS. 8-10). The IC₅₀ was less than 100 nm for the CUTO-3F cells(FIG. 8) and less than 10 nm for the KM12 cells and the MO-91 cells(FIGS. 9 and 10, respectively).

Consistent with the inhibition of cellular proliferation, inhibition ofphosphorylation of the MPRIP-TRKA oncoprotein and ERK1/2 was observed inthe CUTO-3F cells using low doses of the crystalline form (I-HS) (FIG.11), inhibition of the phorphorylation of TPM3-TRKA, pAKT, and pERK1/2in the KM12 cells using low doses of the crystalline form (I-HS) (FIG.12), and inhibition in the phosphorylation of the TEL-TRKC oncoprotein(encoded by ETV6-NTRK3), pAKT, and pERK1/2 in the MO-91 cells using lowdoses of the crystalline form (I-HS) (FIG. 13). Together these data showthat Trk fusion proteins are constitutively active, and regulatecritical downstream signaling pathways, such as MAPK and AKT, and areinhibited by the crystalline form (I-HS). These data also indicate thatthe crystalline form (I-HS) can use used to treat different cancers thatexpress a dysregulated Trk (e.g., a constitutively active form of a Trkprotein (e.g., a Trk fusion protein or a Trk point mutation)).

EXAMPLE 8 The Crystalline Form (I-HS) Successfully Treated a SubjectHaving Undifferentiated Sarcoma

A 41-year-old woman presented with a firm mass in her left groin.Initial imaging was used to confirm a 10-cm mass within the musculatureof her anterior thigh. An open biopsy revealed an undifferentiatedsarcoma. Initial staging scans demonstrated multiple bilateral 4-13 mmpulmonary nodules consistent with metastatic disease. The woman wasenrolled on a phase 2 trial of sorafenib with chemotherapy,pre-operative radiation, and limb-sparing surgery (ClinicalTrials.govnumber NCT02050919). After two weeks of sorafenib administered at 400 mgdaily, the patient received epirubicin at 30 mg/m² daily and ifosfamideat 2,500 mg/m² daily with mesna for three consecutive days, withcontinuation of daily sorafenib. The tumor became progressively morepainful during these five weeks of systemic therapy. During simulationfor pre-operative radiation, extension of the tumor was noted craniallywithin the psoas muscle, precluding the safe administration of effectiveradiation doses due to predicted bowel toxicity. The patient thereforecame off the protocol and proceeded to surgical resection.

Resection of the primary tumor achieved negative margins and review ofthe pathologic specimen confirmed 90% tumor necrosis. A restaging chestCT (shown in FIG. 21A) obtained 9 weeks after initial scans snowedworsening metastatic disease, with the largest nodule now measuring at18 mm. The patient's post-operative course was complicated by apolymicrobial wound infection requiring repeated wound debridement andprolonged antibiotic therapy. Repeat chest CT was obtained beforeresumption of chemotherapy and demonstrated dramatic progression overthe prior 9 weeks, with multiple pulmonary nodules greater than 3 cm,the largest nearly 7 cm, and a large left pleural effusion. Afterplacement of a tunneled pleural drain and initiation of supplementalhome oxygen, the patient received doxorubicin at 75 mg/m² once, whileawaiting enrollment for treatment with the crystalline form (I-HS).

The patient's diagnostic, open tumor biopsy was tested using theFoundationOneHeme panel (Foundation Medicine, Cambridge, Mass.). Thismulti-target comprehensive genomic profiling (CGP) assay using DNA andRNA sequencing of hundreds of cancer-related genes demonstrated thepresence of a gene fusion encoding exons 1-2 of the lamin A/C gene(LMNA) and exons 11-17 of the NTRK1 gene resulting in the LMNA-NTRK1fusion gene (FIG. 14). CGP also showed the loss of the tumor suppressorCDKN2A/B (not shown), but no other known oncogenic mutations.

Subsequently, a break-apart fluorescence in situ hybridization (FISH)assay performed on the patient's tumor sample exhibited a predominantlysingle 3′ NTRK1 (red fluorescence signal) pattern in 64% of tumornuclei, consistent with a genomic alteration involving the NTRK1 genelocus, most likely secondary to a genomic deletion between the two genesgiven the location and orientation of both LMNA and NTRK1 on the largearm of chromosome 1 (FIG. 15). mRNA expression of the novel fusiontranscript from the gene fusion was confirmed by RT-PCR and sequencing(FIG. 16).

A novel proximity ligation assay (PLA) was performed using the patient'stumor sample in order to assess both protein expression and functionalactivity of the fusion oncoprotein. PLAs are unique because they candetect functional signaling complexes between a kinase and one of itsadaptors in situ. In this assay, TRKA complexed with its preferredadaptor, SHC1, which binds to Y496 in the TRKA kinase domain, wasmeasured (FIG. 17). The assay was validated in both human cell lines andformalin-fixed patient-derived tumor xenografts (PDX) tumor samples(FIG. 18). RNAi knockdown of NTRK1 was discovered to disrupt TRKA-SHC1complexes in the CUTO-3 cell line harboring the MPRIP-NTRK1 fusion gene(FIG. 18A-C) as does inhibition with the crystalline form (I-HS) (FIG.18D and 18E). The TRK PLA detects functional signaling complexes in aFFPE tumor sample from a patient derived xenograft (PDX), CULC001,harboring the MPRIP-NTRK1 gene fusions but not the PDX CULC002, whichdoes not harbor a known oncogenic driver mutation (FIG. 18F and 18G).The TRK-SHC PLA can also detect non-oncogenic signaling complexes asshown by a positive signal in a region of peripheral nerve tissue of theCULC001 PDX, where the TRK family of receptors have high expression anactivity mediated by the neurotrophins Application of this assay to thepatient's tumor sample demonstrated robust signaling associated withtumor nuclei, but only a weak signal in the blood vessel (humanendothelial cells express TRKA, consistent with oncogenic signaling bythe LMNA-TRKA oncoprotein (FIG. 19A and B). The TRK-SHC1 PLAdemonstrated a negative result on a tumor sample from an ALK+NSCLCpatient, whereas the ALK-GRB2 PLA was positive (FIG. 20), furtherdemonstrating the ability of this assay to detect oncogenic signaling inhuman tumor samples. The presence of the LMNA-NTRK1 fusion detected byFoundationOneHeme assay and then validated by FISH and RT-PCR combinedwith the evidence of TRKA protein expression and functional activity ofthe TRK pathway in the patient's tumor sample suggests the patient has aTRK-driven cancer suitable for treatment with a TRK-specific inhibitor.

Based on multiple lines of genetic and functional biomarker datasuggesting the presence of a TRK driver oncogene, the patient wasreferred for consideration of enrollment into the phase 1 trial of thecrystalline form (I-HS). A month later, the patient was found eligiblefor the trial and provided written informed consent. The baseline CTscan showed continued tumor progression with multiple large pulmonarymetastases in both lungs, although the pleural effusion had resolvedfollowing placement of the pleural drain (FIG. 21C). On clinicalpresentation the patient had significant exertional dyspnea and required5 L of supplemental oxygen to maintain an oxygen saturation of 90%.Baseline laboratory values were notable for an elevated CA125 tumormarker level (FIG. 22). The patient received an initial dose of 100 mgof the crystalline form (I-HS) three days before the initiation ofcontinuous dosing, followed by the same dose approximately 12 hourslater on the same day, with 48 hours of pharmacokinetic and safetyassessment as per the study protocol. The patient started cycle 1 day 1three days later. The patient was seen weekly for pharmacokinetic andsafety analysis. No drug-related adverse events were noted and thepatient experienced weekly improvement in her exertional dyspnea duringthis 4-week period. The CA125 levels normalized over cycle 1. A CT wasperformed prior to the start of cycle 2 day 1, which demonstrated amarked improvement in multiple pulmonary metastases and was deemed apartial response by RECIST 1.1. A repeat CT was performed prior to cycle3 day 1 and demonstrated an ongoing response and thus confirmed apartial response by RECIST 1.1 (FIG. 21C). Clinically, the patient hadsignificantly improved exertional dyspnea and was no longer requiringsupplemental oxygen with an oxygen saturation of 97% on room air. Afterfour months of dosing, the patient did not have any adverse events thatwere attributed to the crystalline form (I-HS). These data show that thecrystalline form (I-HS) is able to treat an undifferentiated sarcoma ina subject, as well as other cancers that have a dysregulated Trk protein(e.g., a constitutively active form of a Trk protein, e.g., Trk fusionproteins or Trk point mutations).

The LMNA-NTRK1 gene fusion has been previously reported in Spitzoid neviand is constitutively activated when expressed in cells resulting inactivation of ERK1/2, AKT and PLCγ demonstrating its oncogenicity(Taipale et al., Nature Biotech. 31:630-637, 2013). Foundation Medicine(FM) has previously tested 1272 soft tissue sarcoma samples with theFoundationOneHeme CGP test resulting in the detection of 8 NTRK1 orNTRK3 fusions, including the patient described in this case report(Table 15). Notably, 6 of the 8 sarcoma patients with NTRK fusions areunder the age of 25 (Fisher's exact, P-value=4×10⁻⁴) and 4 of the 8 areunder the age of 5 (Fisher's exact, P-value=2×10⁻⁵), indicating anincreased detection rate of NTRK fusions among pediatric patients (4.1%;95% CI 1.8%-9.3%) and particularly those under the age of 5 (14.3%; 95%CI 5.7%-31.5%). Also of interest, one of the gene fusions detectedcombines the majority of the NTRK3 gene (exon 1-17) to the 3′ end of theHOMER2 gene (exons 2-9), which contains a dimerization domain(coiled-coil domain), and therefore represents a 3′ gene fusion eventthat has now been described for multiple other RTK-encoding genes suchas EGFR, AXL, and FGFR3 (Sleijfer et al., Eur. J. Cancer 46:72-83, 2010;Linch et al., Clin. Oncol. 11:187-202, 2014; Rutkowski et al., J. Eur.Acad. Dermatol. Venereol. 25:264-270, 2011).

TABLE 15 Clinical Characteristics and NTRK Fusion Gene Details of SoftTissue Sarcoma Patients Histology 5′ Gene 5′ Last Exon 3′ Gene 3′ FirstExon Gender Age soft tissue sarcoma (nos)(n = 179) LMNA 2 NTRK1 11 F 41soft tissue sarcoma (nos)(n = 179) LMNA 10 NTRK1 11 M 22 soft tissuefibrosarcoma (n = 28) LMA 10 NTRK1 12 M Under 5 soft tissue fibrosarcoma(n = 28) SQSTM1 2 NTRK1 10 F Under 5 soft tissue schwannoma (n = 3) TPM37 NTRK1 10 M Under 5 soft tissue hemangioma (n = 4) ETV6 5 NTRK3 15 FUnder 5 soft tissue solitary fibrous tumor (n = 28) TFC 6 NTRK3 14 M 17soft tissue sarcoma (nos)(n = 179) NTRK3 17 HOMER2 2 F 68Further an experiment was performed to show that the crystalline form(I-HS) specifically inhibits the activity of a Trk kinase. For example,the crystalline form (I-HS) did not inhibit the cellular proliferationof a HCC78 cell line derived from a non-small cell lung cancer thatexpresses the SLC34A2-ROS1 fusion protein (FIG. 23) (Vaishnavi et al.,Nat Med. 19:1469-72, 2013).

Materials and Methods Clinical Trial

NCT02122913 is an ongoing multi-center phase 1 dose-escalation studyevaluating the safety and pharmacokinetics of the crystalline form(I-HS), a selective pan-TRK, in unselected patients with metastatic oradvanced solid tumors without standard therapy options. The study isapproved by Institutional Review Boards at all institutions that enrollpatients, and eligible patients provide written informed consent toparticipate. The crystalline form (I-HS) is provided in 100 mg capsules.Enrolled patients receive escalating doses of the crystalline form(I-HS) according to a modified 3+3 design, and receive the crystallineform (I-HS) daily or twice daily until intolerable toxicity, diseaseprogression, or withdrawal of consent. In patients with measurabledisease, efficacy is assessed per RECIST 1.1 criteria.

Next Generation Sequencing (NGS)

DNA and RNA were extracted and adaptor ligated sequencing libraries werecaptured by solution hybridization using custom bait-sets targeting 405cancer-related genes and 31 frequently rearranged genes by DNA-seq, and265 frequently rearranged genes by RNA-seq (FoundationOne Heme). Allcaptured libraries were sequenced to high depth (Illumina HiSeq) in aCLIA-certified CAP-accredited laboratory (Foundation Medicine),averaging >500× for DNA and >6M unique pairs for RNA. Sequence data fromgDNA and cDNA were mapped to the reference human genome (hg19) andanalyzed through a computational analysis pipeline to call genomicalterations present in the sample, including substitutions, shortinsertions and deletions, rearrangements and copy-number variants.

Fluorescence in situ Hybridization (FISH)

NTRK1 break-apart FISH was performed on 4 micron slides fromformalin-fixed, paraffin embedded (FFPE) tumor samples as previouslydescribed using the Vysis LSI NTRK1 (Cen) SpectrumGreen (Cat #08N43-030)and Vysis LSI NTRK1 (Tel) SpectrumRed (Abbott Molecular, #08N43-030 and08N43-020, respectively) (Vaishnavi et al., Cancer Discov. 5:25-34,2015).

RT-PCR and DNA Sequencing

Reverse trancriptase polymerase chain reaction (RT-PCR) was performed aspreviously described using the forward primer to LMNA (LMNA F1,5′gagggcgagctgcatgat′; SEQ ID NO: 1) (Weisner et al., Nat. Comm. 5:3116,2014) and the reverse primer to NTRK1 (NTRK1 Y490rev,5′cggcgcttgatgtggtgaac′; SEQ ID NO: 2). DNA sequencing of the RT-PCRproduct was performed using Sanger DNA Sequencing at the Pathology Coreat the University of Colorado.

Cell Lines

Informed consent was obtained to derive immortal cell lines from thepatient. CUTO-3 cell line and its derivatives were initiated from themalignant pleural effusion of a stage IV lung adenocarcinoma patientharboring the MPRIP-NTRK1 gene fusion as previously described (Vaishnaviet al., Cancer Discov. 5:25-34, 2015; Davies et al., PLoS One 8:c82236,2013). KM12 and MO-91 have been previously described (Vaishnavi et al.,Nature Med. 19:1469 -1472, 2013; Taipale et al., Nat. Biotech.31:630-637, 2013).

Patient-Derived Xenograft Generation

Informed consent was obtained from the patient to generatepatient-derived murine xenografts. Tumor tissue from an oncogenenegative lung adenocarcinoma patient (CULC001) was cut into 3×3×3 mmpieces that were transferred to DMEM supplemented with 10% fetal bovineserum (FBS) and 200 units/mL penicillin, and 200 ug/mL streptomycin.Tumor pieces were dipped in matrigel (Corning) and inserted intoincisions on each flank of 5 nude mice. Pleural fluid (CULC002) from alung adenocarcinoma patient harboring an MRPIP-NTRK1 gene fusion wascentrifuged and the resulting cell pellet was suspended in 5 ml ACKbuffer (Lonza) for 2 min allowing for the complete lysis of red bloodcells. Lysis was halted by the addition of 20 mL PBS and centrifugingthe samples. The pellet was washed twice PBS prior to being suspended inDMEM supplemented media as above. 100 μl of cells (1×10⁶ per flank)suspended in a 1:1 mix of DMEM and matrigel (BD) were injectedsubcutaneously into the flanks of 5 nude mice. Propagation andmaintenance of resulting xenografts was previously described (Keysar etal., Mol. Oncol. 7:776-790, 2013).

Proximity Ligation Assays

Cells were seeded onto glass coverslips (in a 48 well plate) or chamberslides at 25-75 k cells/well. Cells were treated with the indicateddoses and times then fixed for 15 minutes by shaking at room temperaturein 4% paraformaldehyde. The cells were rinsed twice in PBS, and then theDuolink® in situ PLA® kit from SigmaAldrich in mouse/rabbit (Red) wasused according to the manufacturer's protocol (catalog #DUO92101). Theantibody concentrations were optimized using immunofluorescence prior toPLA experiments. The FFPE tissue PLAs from mice or patients wereprepared as described in histology. Additionally, samples were treatedwith 300 mM glycine for 15 minutes prior to the blocking step, otherwisethe assay was performed according to the manufacturer's protocol. Thecells were mounted using Prolong® gold anti-fade reagent (with DAPI) andcured overnight prior to imaging. The images were either taken on aNikon standard inverted fluorescent microscope at 40×, or on the 31Marianas spinning disc confocal in the University of Colorado AnschutzMedical Campus Advance Light Microscopy Core at 40× or 100×. Thefollowing antibodies were used: TRK (C17F1) and ALK (D5F3) from CellSignaling, SHC1 from Novus, and Grb2 (610111) from BD.

Proliferation Assays

All proliferation assays were performed in media supplemented with 5%FBS as previously described using Cell Titer 96 MTS (Promega) (Bouhanaet al., EORTC-NCI-AACR 26^(th) Symposium on Molecular Targets and CancerTherapeutics, Barcelona, Spain 2014). Cells were seeded 500-2000cells/well and treated for 72 hours at the drug concentrations describedon each graph. Each assay was performed in triplicate in at least 3independent biological replicates. Data were plotted and IC50 valuescalculated using GraphPad software.

Immunoblotting

Immunoblotting was performed as previously described (Vaishnavi et al.,Nature Med. 19:1469-1472, 2013). Briefly, cells were lysed in RIPAbuffer with Halt Protease and Phosphatase Inhibitor Cocktail (ThermoScientific) and diluted in loading buffer (LI-COR Biosciences). Themembranes were scanned and analyzed using the Odyssey Imaging System andsoftware (LI-COR). The following antibodies were used from CellSignaling: pTRK Y490 (rabbit polyclonal, #9141), pERK1/2 XP T202/Y204(#9101), total ERK1/2, pAKT S473 (rabbit mAb, #4060), and total AKTmouse clone D3A7 (#2920). TRK (C-14) rabbit polyclonal antibody waspurchased from Santa Cruz Biotechnology. GAPDH (MAB374) and pTYR (4G10)are from Millipore.

Statistical Analysis

Confidence intervals for the detection rate of NTRK fusions in samplesfrom sarcoma patients were calculated using the 1-sample proportionstest. The disease histology classification was based on the FoundationMedicine disease ontology. The enrichment of NTRK fusions in youngerpatient groups was tested using Fisher's Exact Test. All statisticaltesting was performed in R v 3.1.3.

EXAMPLE 9 Clinical Safety and Activity from a Phase 1 Study ofCrystalline Form (I-HS), a Selective TRKA/B/C Inhibitor, in Solid-TumorPatients with NTRK Gene Fusions Methods

In this on-going open-label, multicenter, 3+3 dose escalation Phase Istudy of crystalline form (I-HS), 23 patients with solid tumorsrefractory to standard therapy, normal hematopoietic and major organfunction have been enrolled. Crystalline form (I-HS) was administeredorally as a single dose, followed by QD or BID doses for continuous28-day cycles. Response is measured by RECIST Criteria, version 1.1.Serum is collected for pharmacokinetic analysis on Cycle 1 Day 1 and Day8. Safety information is collected on all patients and the definition ofdose-limiting toxicity applies to adverse events regardless ofrelationship to investigational product.

Results

To date, 23 patients were treated at each of the first five dose levelsranging from 50 mg QD-150 mg BID. Crystalline form (I-HS) has been welltolerated; the MTD has not been reached and the most common adverseevents are Grade 1 and 2 fatigue (35%), dizziness (26%) and anemia(22%). Two patients had dose limiting toxicities (elevated AST, grade 3(Dose Level 150 mg BID) and delirium, grade 3 unrelated (Dose Level 100mg BID)).

PK analysis showed maximum plasma concentrations of crystalline form(I-HS) were reached 30-60 minutes following dosing and exposureincreased in approximate proportion with dose. The unbound drug levelsof crystalline form (I-HS) appear sufficient for approximately 98%inhibition of TRKA/B/C at peak concentrations at all dose levels.

Three of the 23 patients harbored NTRK-fusions and were treated ateither 100 or 150 mg BID. These patients achieved a partial response: anundifferentiated sarcoma with an LMNA-NTRK1 fusion (59% decrease; 7cycles+), a c-kit-negative GI Stromal Tumor (GIST) with an ETV6-NTRK3fusion (30% decrease; 2 cycles+), and a mammary analogue secretorycarcinoma with an ETV6-NTRK3 fusion (64% decrease; 2 cycles+). Thesedata are supported by in vivo tumor growth inhibition and regression inxenograft mouse models of TRK-fusions.

Conclusions

Crystalline form (I-HS) has been well tolerated and has sufficientsystemic exposure for robust inhibition of the NTRK-fusions as evidencedby pharmacokinetic drug levels, and the ongoing clinical responsesobserved in the 3 NTRK-fusion patients enrolled in this study. Thesedata further validate this molecular target as an oncogenic driveracross diverse tumor histologies.

EXAMPLE 10 Comparison of Crystalline Form (I-HS) and the AmorphousSulfate Salt

Various experiments were performed to compare properties of amorphous(S)—N-(5-((R)-2-(2,5-difluorophenyl)-pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehydrogen sulfate and crystalline form (I-HS). These studies includeimpurity profiles, stability, flow properties and hygroscopicity. In thefollowing studies, two lots of amorphous material (AM(HS)1 and AM(HS)2)were compared to a single lot of crystalline form (I-HS). AM(HS)1 andAM(HS)2 were prepared as described in Example 3. Crystalline form (I-HS)was prepared as described in Example 2.

Methods Residual Solvents

Solutions of AM(HS)1, AM(HS)2, and crystalline form (I-HS) were analyzedusing GC-MS headspace analysis.

Thermogravimetric Analysis (TGA)

Samples placed on platinum pans and subjected to 10° C./minute to 300°C.

Differential Scanning Calorimetry (DSC)

Samples were placed in crimped aluminum crucibles with a pin-hole in thelid and subjected to 10° C./minute to 250° C. under nitrogen.

X-Ray Diffraction (XRD)

Cu Kα Radiation at 44 kV, 40 mA through a Ni filter with a divergenceslit of ⅔°, Divergence H. L. slit of 10 mm, Scatter slit set to “Auto”(the scatter slit is determined by the computer/instrument), Receivingslit of 0.3 mm. Continuous scan from 3° to 40° 2θ at 2°/min; samplingwidth (step size) of 0.02°/second, step time of 0.4 point/second.Samples were rotated on a plane parallel to sample surface at 60 rpm.

Polarized Light Microscopy (PLM)

Samples were placed on a glass microscope slide, bathed in low-viscosityoil and covered with a glass coverslip. Examined under 20× objectivelens with cross-polarized lenses and a 530 nm cut-off filter. Imagingdone with a PAX-It camera and processed by PAX-It software.

Dynamic Vapor Sorption (DVS)

-   -   1. The hygroscopicity was studied at 25° C. using an IGAsorp        analyzer.    -   2. About 15 mg of sample was placed in a tared mesh sample        holder at an initial ambient room humidity setting of ˜35%.    -   3. A total wet/dry nitrogen flow rate of 250 cc/min is used        throughout the study.    -   4. Solids were studied by performing one full cycle of the        following program: 60 minutes of drying at 40° C. under dry N₂,        followed by settings of 0, 10, 20, 30, 40, 50, 60, 70, 80, 90        and 95% RH, with exposure time at each humidity set point        dependent upon 99.5% confidence in the F1 fit model or 60        minutes. The maximum time allowed at any one humidity set point        was 120 min. The sample was maintained under dry N₂ after the        cycle was completed.        Percent weight gain was calculated based on the dry weight        basis.

Part One: Physical Characterization of Crystalline Form (I-HS)

AM(HS)1, AM(HS)2, and crystalline form (I-HS) were characterized byappearance, residual solvents, Thermogravimetric Analysis (TGA), KarlFischer water content (KF), Differential Scanning Calorimetry (DSC),X-Ray Diffraction (XRD), Polarized Light Microscopy (PLM) andhygroscopicity by Dynamic Vapor Sorption (DVS). The data for thephysical characterization of the three lots can be found in the Tables16-19 and FIGS. 24-38.

TABLE 16 Appearance Compound Appearance Munsell # AM(HS)1 Yellow Powderfree of aggregates 7.5Y 9/10 AM(HS)2 Yellow Powder free of aggregates7.5Y 9/10 Crystalline I-HS Orange Powder free of aggregates 2.5YR 7/10

TABLE 17 Residual Solvents Solvents (ppm) Methyl t-butyl Methyl ethylCompound Ethanol Methanol ether Heptane Tetrahydrofuran ketone AM(HS)1not 1075 not tested not tested not tested not tested detected AM(HS)2174 7369 not tested not tested not tested not tested Crystalline I-HS4783 not not detected not not detected 3046 tested detected

TABLE 18 Thermalgravimetric and Karl Fischer (water content) AnalysesThermogravimetric Analysis Start Stop Change KF Compound (° C.) (° C.)(%) (w/w %) AM(HS)1 24.78 111.94 3.78 2.00 111.94 186.94 3.09 AM(HS)231.53 100.21 1.94 0.88 100.21 174.86 3.07 Crystalline I-HS 23.77 218.586.01 0.28

TABLE 19 Differential Scanning Calorimetry Analyses DifferentialScanning Calorimetry Start Onset Maximum Stop ΔH Compound Type (° C.) (°C.) (° C.) (° C.) (J/g) AM(HS)1 endotherm 28.7 29.9 68.3 115.8 153.7endotherm 124.1 126.1 152.8 191.0 58.3 endotherm 204.7 205.6 211.2 220.17.5 AM(HS)2 endotherm 29.6 29.8 70.7 107.2 70.0 endotherm 113.7 116.0140.3 167.0 32.7 endotherm 205.6 207.2 213.8 221.3 4.8 Crystalline I-HSendotherm 181.9 193.7 204.6 213.8 98.9

Physical Characterization Conclusion

API forms AM(HS)1 and AM(HS)2 are amorphous with no birefringence bypolarized light microscopy and the XRPD pattern is also distinctlyamorphous for both lots with no discernible x-ray diffraction peaks. TheTGA for the amorphous compounds shows step-wise weight losscorresponding with endothermic events observed in differential scanningcalorimetry. The first two endothermic events are quite broad and mayindicate an evaporation and/or de-solvation. The last endothermic eventoccurs at the approximate temperature of the melt observed incrystalline material. Both amorphous lots are rather hygroscopic withsignificant hysteresis upon desorption. AM(HS)1 gained greater than 13%of its original mass at 80% RH. Likewise, AM(HS)2 gained nearly 12% ofits starting mass at 80% RH. Post-DVS XRPD indicates that there was noform change during the dynamic vapor sorption however it was observedthat the powder deliquesced in the sample holder making removal of thedeliquesced powder from the sample holder difficult.

Crystalline form (I-HS) is crystalline in nature with many diffractionpeaks by x-ray diffraction and sincere birefringence by polarized lightmicroscopy in its agglomerate-like morphology. Crystalline form (I-HS)shows a thermogravimetric weight loss of 6% corresponding with anendothermic melt onset occurring at 193.7° C. Crystalline form (I-HS) isnot hygroscopic, and gained only 1% of its starting mass at 80% RH.

Part Two: Powder Properties of Crystalline Form (I-HS)

The following studies compared the crystalline hydrogen sulfate saltwith the amorphous sulfate salt powder including a study of each form'sflow properties which are important for manufacturing a solid oraldosage form such as a tablet or capsule. Work performed includes bulkdensity, tapped density, angle of repose, and compression profiles.

Blends

Blends were created according to the formulations presented in Tables 20and 21. These blends are typical of tablet formulations that could bemanufactured as a direct compression or roller compaction based tabletor formulated capsule. First API (i.e. either AM(HS) or crystalline form(I-HS)), microcrystalline cellulose (MCC), and either starch or lactosewere added to a 30 cc amber glass bottle and blended on the TURBULA®Shaker-mixer at 25 rpm for 3 minutes. Then the remaining excipients wereadded to the bottle and blended on the TURBULA® at 25 rpm for anadditional 3 minutes.

TABLE 20 Tablet Formulation with 2:1 MCC:Lactose, 50% Drug Load TargetCrystalline Amorphous Component Grade Purpose Percentage Mass(g) ActualMass(g) Actual Mass(g) API NA Drug Substance 50.00 2.500 2.5004 2.5009MCC PH102 NF diluent 30.30 1.515 1.5152 1.5153 Lactose Fast-Flo 316diluent 15.20 0.760 0.7603 0.7602 Croscarmellose Sodium Ac-Di-Soldisintegrant 3.00 0.150 0.1503 0.1498 Silicon Dioxide Cabosil glidant1.00 0.050 0.0502 0.0497 Mg. Stearate USP-NF lubricant 0.50 0.025 0.02480.0254 Total 100.00 5.000 5.0012 5.0013

TABLE 21 Tablet Formulation with 1:1 MCC:Starch, 50% Drug Load TargetCrystalline Amorphous Component Grade Purpose Percentage Mass(g) ActualMass(g) Actual Mass(g) API N/A Drug Substance 50.00 2.500 2.4994 2.4998MCC PH102 NF diluent 22.75 1.138 1.1385 1.1387 Pre-gelatinized starchStarCap 1500 diluent 22.75 1.138 1.1385 1.1376 Croscarmellose SodiumAc-Di-Sol disintegrant 3.00 0.150 0.1507 0.1503 Silicon Dioxide Cabosilglidant 1.00 0.050 0.0500 0.0498 Mg. Stearate USP-NF lubricant 0.500.025 0.0252 0.0250 Total 100.00 5.000 5.0023 5.0012

Angle of Repose

The angle of repose is the angle formed by the horizontal base of thesurface and the edge of a cone-like pile of granules. It is calculatedfrom the following equation:

$\theta = {\tan^{- 1}\left( \frac{h}{r} \right)}$

The angle of repose was measured by slowly pouring approximately 1 g ofsample through a funnel with a 3/16″ inner diameter of the outlet. Thepowder then fell 1 11/16″ to land on the surface of an overturnedcrystallization dish on which a pile of powder formed. A picture wastaken of the pile after addition of all of the material. The anglebetween the dish surface and the surface of the pile was measure via aprotractor on the pictures. Care was taken to replicate the samepositions and fall distances in the set up between different samples.

Bulk and Tap Densities

The powder was added to a pre-weighed 10 mL graduated cylinder through afunnel that was not in direct contact with the graduated cylinder toavoid transference of vibrations. Powder was added until a 10 mL volumewas reached and then the graduated cylinder with powder was weighed. Thebulk density was calculated by

${{Bulk}\mspace{14mu} {Density}} = {\frac{\left( {{{Mass}\mspace{14mu} {of}\mspace{14mu} {cylinder}} + {powder}} \right) - {{Mass}\mspace{14mu} {of}\mspace{14mu} {empty}\mspace{14mu} {cylinder}}}{10\mspace{14mu} {mL}}.}$

The same sample in the graduated cylinder was tapped for the followingsequence: 100, 150, 250, 250 taps. The volume was measured after eachinterval. The tapped density was calculated by

${{Tapped}\mspace{14mu} {Density}} = {\frac{\left( {{{Mass}\mspace{14mu} {of}\mspace{14mu} {cylinder}} + {powder}} \right) - {{Mass}\mspace{14mu} {of}\mspace{14mu} {empty}\mspace{14mu} {cylinder}}}{{Volume}\mspace{14mu} {after}\mspace{14mu} 750\mspace{14mu} {total}\mspace{20mu} {taps}}.}$

The Can's Index was calculated according to the following equation:

${C\; I} = {\frac{{\rho \;}_{tap} - \rho_{bulk}}{{\rho \;}_{tap}} \times 100}$

The Hausner Ratio was calculated with the following equation:

${H\; R} = \frac{{\rho \;}_{tap}}{{\rho \;}_{bulk}}$

Compression Profiles

Compression profiles were generated by creating 5/16″ diameter, roundtablets at five different compression pressures for each blend. Thepress settings were 1 second dwell time and 15% pump speed. Tablets werecreated for all four powder blends using compression forces of 700 kg,1000 kg, 1500 kg, and 2000 kg. The highest compression force wasselected based on the results of the previous tablets. Tablet mass,dimensions, and rupture force were then measured. These data wereplotted using a template resulting in a plot of compression pressure vs.tensile strength (FIG. 39).

Results

TABLE 22 Reference values Angle of Hausner Compressibility FlowCharacter Repose Ratio Index (%) Excellent 25-30° 1.00-1.11 ≦10 Good31-35° 1.12-1.18 11-15 Fair 36-40° 1.19-1.25 16-20 Passable 41-45°1.26-1.34 21-25 Poor 46-55° 1.35-1.45 26-31 Very Poor 56-65° 1.46-1.5932-27 Very, Very Poor ≧66° ≧1.60 ≧38

Results are presented in Tables 23 and 24 and FIG. 39. According to theU.S. Pharmacopeial Convention (USP), all samples fall into the passableor poor category for flow as measured by angle of repose. Larger anglesindicate worse flow. The Can's Index (Compressibility Index) and HausnerRatio fall between passable and very poor according to USP. Thecrystalline API is noticeably different from the amorphous API and thesedifferences are present in all formulation blends irrespective of thecontent of amorphous or crystalline API. For the crystalline API, theHausner Ratio and the Carr's Index indicate flow properties are“Passable”. The amorphous API has considerably worse flow properties,being categorized as “Very Poor” for both the Hausner ration and Carr'sIndex. See Table 22 for the relevant USP tables.

When creating tablets for the compression profiles, the crystalline APIblends produced multiple tablets that had breakage upon ejection fromthe tooling. The amorphous API blends seemed to produce visually bettertablets, i.e. little to no breakage.

TABLE 23 Angle of Repose Sample θ(°) φ(°) Average(°) L-ARR10-118 50.2850.28 50.28 AR00457470-33 47.79 43.6 45.70 2:1 MCC:Lactose L-ARR10-11845.21 41.81 43.51 2:1 MCC:Lactose AR00457470-33 41.01 42.64 41.83 1:1MCC:Starch L-ARR10-118 39.52 41.01 40.27 1:1 MCC:Starch AR00457470-3340.44 48.59 44.52 *θ and φ are the angles on either side of the pyramid(2D).

TABLE 24 Bulk and Tap Densities Bulk Tapped Density Density Carr HausnerSample (mg/mL) (mg/mL) Index Ratio L-ARR10-118 594.8 762.6 22% 1.28AR00457470-33 423.6 622.9 32% 1.47 2:1 MCC:Lactose L-ARR10-118 435.3621.9 30% 1.43 2:1 MCC:Lactose AR00457470- 408.1 583.0 30% 1.43 33 1:1MCC:Starch L-ARR10-118 437.9 625.5 30% 1.43 1:1 MCC:Starch AR00457470-411.4 605.0 32% 1.47 33

Powder Properties Conclusion

By the angle of repose the crystalline form (I-HS) formulation blends,amorphous API and amorphous API formulation blends tested have “VeryPoor” flow characteristics. However, by Carr's Index and Hausner Ratiothe crystalline API, crystalline form (I-HS), has “passable” flowcharacteristics. The significantly better flow properties here are anadvantage for solid oral dosage form development and manufacturing.There was also not a large difference in the compression profile of bothblends with both lots of powder. This is an indication that(5)-N-(5-((R)-2-(2,5-difluorophenyl)-pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehydrogen sulfate whether amorphous or crystalline did not positively nornegatively affect the flow of the blends at a 50% drug load.

Part Three: Stability of AM(HS)1 and Crystalline Form (I-HS)

Aliquots of powder of AM(HS)1 and crystalline form (I-HS) were placedinto non-capped (open) 20 mL scintillation vials and the vials placedinto an LDPE bag put into a stability chamber maintained at 40° C./75%RH for five weeks. Upon removal from the chamber, the samples werephysically characterized by appearance, KF, TGA, DSC, XRD and PLM. Thesamples were also analyzed by HPLC for chromatographic purity, chiralintegrity and potency. Where applicable, the data presented in thestability portion also includes the original T=0 data for comparisonpurposes. The data can be seen in Tables 25-29 and FIGS. 40-44.

TABLE 25 Appearance of Stability Samples Time point Munsell CompoundCondition (wks.) Appearance # AM(HS)1 NA 0 Yellow Powder 7.5Y free ofaggregates 9/10 AM(HS)1 40° C./75% 5 Orange Powder 2.5YR RH someaggregates 7/10 Crystalline form NA 0 Orange Powder 2.5YR (I-HS) free ofaggregates 7/10 Crystalline form 40° C./75% 5 Orange Powder 2.5YR (I-HS)RH free of aggregates 7/10

TABLE 26 TGA and KF of Stability Samples Time Thermogravimetric AnalysisKF point Start Stop Change (w/w Compound Condition (wks.) (° C.) (° C.)(%) %) AM(HS)1 NA 0 24.78 111.94 3.78 2.00 111.94 186.94 3.09 AM(HS)140° C./ 5 36.27 217.16 5.32 0.56 75% RH Crystalline form NA 0 23.77218.58 6.01 0.28 (I-HS) Crystalline form 40° C./ 5 36.66 217.87 6.000.21 (I-HS) 75% RH

TABLE 27 DSC of Stability Samples Time Differential Scanning Calorimetrypoint Start Onset Maximum Stop ΔH Cmpd Condition (wks.) Type (° C.) (°C.) (° C.) (° C.) (J/g) AM(HS)1 NA 0 endotherm 28.7 29.9 68.3 115.8153.7 endotherm 124.1 126.1 152.8 191.0 58.3 endotherm 204.7 205.6 211.2220.1 7.5 AM(HS)1 40° C./75% 5 endotherm 182.7 196.7 206.3 217.7 95.9Crystalline NA 0 endotherm 181.9 193.7 204.6 213.8 98.9 form (I-HS)Crystalline 40° C./75% 5 endotherm 181.3 193.4 204.1 214.1 99.4 form(I-HS) RH

TABLE 28 HPLC Data of Stability Samples Time Total Chiral pointImpurities Assay Potency Compound Condition (wks.) (%) (%) (%) AM(HS)1NA 0 1.10 79.5 99.6 AM(HS)1 40° C./75% 5 1.16 80.3 99.6 RH Crystallineform NA 0 0.14 79.4 99.6 (I-HS) Crystalline form 40° C./75% 5 0.07 79.699.6 (I-HS) RH

TABLE 29 HPLC Data: Peak Area Percent by RRT of Stability Samples Timepoint RRT Sample Condition (wks.) 0.793 0.863 0.981 1.000 1.535 AM(HS)1NA 0 0.00 0.98 0.12 98.89 0.00 40° C./75% RH 5 0.00 1.04 0.12 98.83 0.00Crystalline form NA 0 0.07 0.00 0.00 99.86 0.07 (I-HS) 40° C./75% RH 50.07 0.00 0.00 99.93 0.00

Stability Conclusions

Amorphous compound AM(HS)1 was not stable in humidified conditions andtended to crystallize over a period of time. This was evidenced by thedeliquescence of samples left in humidity chambers and the changedappearance both by the eye and polarized light microscopy (data notshown). The amorphous material goes from a free flowing yellow powder toan orange agglomerated non-free flowing powder. The polarized lightmicroscopy, XRPD, DSC, TGA and KF of the amorphous API also changedsignificantly over the course of the accelerated stability study tobecome the same polymorphic form as the crystalline form (I-HS). TheXRPD pattern of the amorphous compound AM(HS)1 transforms into the XRPDpattern of crystalline form (I-HS) over the course of the acceleratedstability study. The polarized light microscopy goes fromnon-birefringent to birefringent under cross-polarized light which isindicative of a change from amorphous to crystalline API. The DSC at t=0has two endothermic events with melt maximums at 68.3° C. and 152.8° C.that disappear at the t=5 week time point. There is only a singleendothermic event remaining for the amorphous material with a meltmaximum at 206° C. This melt maximum matches the thermographic profileof the crystalline API. The TGA profile of the amorphous material at t=5weeks also changed to match the profile and weight loss of thecrystalline API. Crystalline form (I-HS) exhibited no hygroscopicity norany change in color, morphology or crystallinity after storage underaccelerated conditions.

The API chemical purity did not change significantly over the course ofthe stability study for either the AM(HS)1 or crystalline form (I-HS).The impurity profiles of the amorphous and crystalline form (I-HS) are,however, significantly different. The amorphous material containssignificantly higher levels of impurities (Tables 22 and 23) versus thecrystalline form (I-HS). The reduced impurities in the crystalline form(I-HS) vs. the amorphous AM(HS)1 at relative retention times (RRT) 0.863(0.00% vs. 0.98%) and 1.535 (0.00% vs. 0.12%) is believed to be due tothe isolation of crystalline form (I-HS) via a crystallization processthat rejects these impurities and is superior to the method of isolationfor the amorphous AM(HS)1. The amorphous AM(HS)1 isolation process doesnot appear to reject these impurities as efficiently.

Overall Summary of Study

-   -   1. The crystalline form (I-HS) has better flow properties vs.        the amorphous form AM(HS). The differences in flow properties        will improve development of a solid oral dosage form crystalline        form (I-HS) vs. the AM(HS).    -   2. The stability study in an LDPE bag at 40° C./75% RH for five        weeks did not show significant changes in chemical impurity        levels for either forms of the compound. It did, however, reveal        that crystalline form (I-HS) did not have a significant change        in its physicochemical properties over the course of the study.        In contrast, AM(HS), converted into a crystalline form        substantially similar to crystalline form (I-HS) by XRPD, DSC,        TGA, KF and polarized light microscopy. Additionally, AM(HS)        changed to an agglomerated powder with reduced flow properties        over the course of the stability testing. A change in the        amorphous AM(HS) properties to a crystalline material and/or an        agglomerated powder with reduced flow ability on storage of        AM(HS) would make it impossible to manufacture a solid oral        dosage form for patient use based on the amorphous compound.    -   3. AM(HS) deliquesced when exposed to humidity. This would        require significant handling precautions during storage and        manufacture to prevent this occurrence whereas crystalline form        (I-HS) requires no such precautions during manufacture of        crystalline form (I-HS) and any solid oral dosage product        prepared using crystalline form (I-HS).    -   4. Crystalline form (I-HS) provides a significantly improved        impurity profile as compared to AM(HS). The ability to control        an impurity profile is important for patient safety and required        by Regulatory agencies.

EXAMPLE 11 The Crystalline Form (I-HS) Decreases the Growth of TumorsCharacterized as Expressing a Trk Fusion Protein

A set of experiments were performed to determine whether the crystallineform (I-HS) would inhibit the growth of three different xenograph(human) tumors in mice, with each xenograph tumor being derived from acancer cell line. The three different cancer cell lines, the CUTO-3Fcell line, the KM12 cell line, and the MO-91 cell line, each express adifferent Trk gene fusion. As described in Example 7, the CUTO-3F cellline is derived from a patient with lung adenocarcinoma harboring theMPRIP-NTRK1 gene fusion, the KM12 cell line is a colorectal cancer cellline harboring the TPM3-NTRK1 fusion (Vaishnavi et al., Nature Med.19:1469-1472, 2013), and the MO-91 cell line is derived from an acutemyeloid leukemia patient harboring the ETV6-NTRK3 fusion (Taipale etal., Nature Biotech. 31:630-637, 2013). Following implantation of one ofthese three different xenograph (human) tumors in mice, the change inthe volume of each tumor was monitored. The resulting mice were lefttreated with vehicle or were orally administered a daily dose of 60mg/kg or 200 mg/kg of crystalline form (I-HS) (FIGS. 45-47,respectively) following implantation of the xenograft.

These data show that administration of the crystalline form (I-HS) isable to effectively halt the growth, or decrease the rate of growth, ofhuman tumors characterized by expression of an oncogenic Trk fusionprotein in a mammal.

While the foregoing specification teaches the principles of the presentinvention, with examples provided for the purpose of illustration, itwill be understood that the practice of the invention encompasses all ofthe usual variations, adaptations and/or modifications as come withinthe scope of the following claims and their equivalents.

REFERENCES

-   -   1. Wiesner et al., Nature Comm. 5:3116, 2014.    -   2. Vaishnavi et al., Nature Med. 19:1469-1472, 2013.    -   3. Greco et al., Mol. Cell. Endocrinol. 28:321, 2010.    -   4. Kim et al., PloS ONE 9(3):e91940, 2014.    -   5. Vaishnavi et al., Nature Med. 19:1469-1472, 2013.    -   6. Fernandez-Cuesta et al., “Cross-entity mutation analysis of        lung neuroendocrine tumors sheds light into their molecular        origin and identifies new therapeutic targets,” AACR Annual        Meeting 2014, Abstract, April 2014.    -   7. Stransky et al., Nature Comm. 5:4846, 2014.    -   8. Ross et al., Oncologist 19:235-242, 2014.    -   9. Doebele et al., J. Clin. Oncol. 32:5s, 2014.    -   10. Jones et al., Nature Genetics 45:927-932, 2013.    -   11. Wu et al., Nature Genetics 46:444-450, 2014.    -   12. WO 2013/059740    -   13. Zheng et al., “Anchored multiplex PCR for targeted        next-generation sequencing,” Nature Med., published online on        Nov. 10, 2014.    -   14. Caria et al., Cancer Genet. Cytogenet. 203:21-29, 2010.    -   15. Frattini et al., Nature Genet. 45:1141-1149, 2013.    -   16. Martin-Zanca et al., Nature 319:743, 1986.    -   17. Meyer et al., Leukemia 21: 2171-2180, 2007.    -   18. Reuther et al., Mol. Cell. Biol. 20:8655-8666, 2000.    -   19. Marchetti et al., Human Mutation 29(5):609-616, 2008.    -   20. Tacconelli et al., Cancer Cell 6:347, 2004.    -   21. Walch et al., Clin. Exp. Metastasis 17: 307-314, 1999.    -   22. Papatsoris et al., Expert Opin. Invest. Drugs 16(3):303-309,        2007.    -   23. Van Noesel et al., Gene 325: 1-15, 2004.    -   24. Zhang et al., Oncology Reports 14: 161-171, 2005.    -   25. Truzzi et al., J. Invest. Dermatol. 128(8):2031, 2008.    -   26. Kolokythas et al., J. Oral Maxillofacial Surgery        68(6):1290-1295, 2010.    -   27. Ni et al., Asian Pacific Journal of Cancer Prevention        13:1511, 2012.

What is claimed:
 1. A crystalline form (I-HS) having the formula:


2. The crystalline form according to claim 1, characterized by havingXRPD diffraction peaks (2θ degrees) at 18.4±0.2, 20.7±0.2, 23.1±0.2, and24.0±0.2.
 3. The crystalline form according to claim 1, characterized byhaving XRPD diffraction peaks (2θ degrees) at 10.7±0.2, 18.4±0.2,20.7±0.2, 23.1±0.2, and 24.0±0.2.
 4. The crystalline form according toclaim 1, characterized by having XRPD diffraction peaks (2θ degrees) at10.7±0.2, 18.4±0.2, 19.2±0.2, 20.2±0.2, 20.7±0.2, 21.5±0.2, 23.1±0.2,and 24.0±0.2.
 5. The crystalline form according to claim 1,characterized by having XRPD diffraction peaks (2θ degrees) at 10.7±0.2,15.3±0.2, 16.5±0.2, 18.4±0.2, 19.2±0.2, 19.9±0.2, 20.2±0.2, 20.7±0.2,21.5±0.2, 22.1±0.2, 23.1±0.2, 24.0±0.2. 24.4±0.2, 25.6±0.2, 26.5±0.2,27.6±0.2, 28.2±0.2, 28.7±0.2, 30.8±0.2, and 38.5±0.2.
 6. The crystallineform according to claim 1, wherein the crystalline form has XRPD patternsubstantially as shown in FIG.
 29. 7. The crystalline form according toclaim 1, wherein the crystalline form exhibits an onset to maximum ofabout 193 to about 205° Celsius, as measured by differential scanningcalorimetry.
 8. The crystalline form according to claim 1, wherein thecrystalline form exhibits a heat of melting of about 2.415 mW, asmeasured by differential scanning calorimetry.
 9. The crystalline formaccording to claim 1, wherein the crystalline form has a DSC thermogramsubstantially as shown in FIG.
 26. 10. The crystalline form according toclaim 1, wherein the crystalline form is non-hygroscopic.
 11. Apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a crystalline form according to claim
 1. 12. Apharmaceutical composition made by mixing a crystalline form accordingto claim 1 and a pharmaceutically acceptable carrier.
 13. A process formaking a pharmaceutical composition comprising mixing a crystalline formaccording to claim 1 and a pharmaceutically acceptable carrier.
 14. Amethod of treating a disorder selected from the group consisting ofcancer, pain, inflammation, neurodegenerative disease or Trypanosomacruzi infection, the method comprising administering to a subject inneed thereof a therapeutically effective amount of a crystalline formaccording to claim
 1. 15. A method of treating a cancer mediated by aTrk kinase in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of acrystalline form according to claim
 1. 16. The method of claim 15,wherein the cancer is mediated by TrkA.
 17. The method of claim 15,wherein the cancer is mediated by TrkB.
 18. The method of claim 15,wherein the cancer is mediated by TrkA and TrkB.
 19. A method oftreating a patient diagnosed or identified as having a Trk-associatedcancer, the method comprising administering to the subject atherapeutically effective amount of a crystalline form according toclaim
 1. 20. A method for treating cancer in a subject in need thereof,the method comprising: (a) determining if the cancer is associated withone or more of overexpression, activation, amplification, and mutationof a Trk kinase; and (b) if the cancer is determined to be associatedwith one or more of overexpression, activation, amplification, andmutation of a Trk kinase, administering to the subject a therapeuticallyeffective amount of a crystalline form according to claim
 1. 21. Amethod for treating cancer in a subject in need thereof, the methodcomprising: (a) determining if the cancer is mediated by a Trk kinase;and (b) if the cancer is determined to be mediated by a Trk kinase,administering to the subject a therapeutically effective amount of acrystalline form according to claim
 1. 22. A method of treating asubject comprising: (a) performing an assay on a sample obtained fromthe subject to determine whether the subject has dysregulation of a NTRKgene, a Trk protein, or expression or level of the same; and (b)administering to a subject determined to have dysregulation of a NTRKgene, a Trk protein, or expression or activity, or level of the same atherapeutically effective amount of a crystalline form according toclaim
 1. 23. The method of claim 19 or 22, wherein the dysregulation ofa NTRK gene, a Trk protein, or expression or level of the same is achromosome translation that results in the translation of a Trk fusionprotein.
 24. The method of claim 23, wherein the Trk fusion protein isselected from the group consisting of: TP53-TrkA, LMNA-TrkA, CD74-TrkA,TFG-TrkA, TPM3-TrkA, NFASC-TrkA, BCAN-TrkA, MPRIP-TrkA, TPR-TrkA,RFWD2-TrkA, IRF2BP2-TrkA, SQSTM1-TrkA, SSBP2-TrkA, RABGAP1L-TrkA,C18ORF8-TrkA, RNF213-TrkA, TBC1D22A-TrkA, C20ORF112-TrkA, DNER-TrkA,ARHGEF2-TrkA, CHTOP-TrkA, PPL-TrkA, PLEKHA6-TrkA, PEAR1-TrkA,MRPL24-TrkA, MDM4-TrkA, LRRC71-TrkA, GRIPAP1-TrkA, EPS15-TrkA,DYNC2H1-TrkA, CEL-TrkA, EPHB2-TrkA, TGF-TrkA, NACC2-TrkB, QKI-TrkB,AFAP1-TrkB, PAN3-TrkB, SQSTM1-TrkB, TRIM24-TrkB, VCL-TrkB, AGBL4-TrkB,DAB2IP-TrkB, ETV6-TrkC, BTBD1-TrkC, LYN-TrkC, RBPMS-TrkC, EML4-TrkC,HOMER2-TrkC, TFG-TrkC, FAT1-TrkC, and TEL-TrkC.
 25. The method of claim19 or 23, wherein the dyregulation of a NTRK gene, a Trk protein, orexpression or activity of the same is one or more point mutation in thegene.
 26. The method of claim 25, wherein the NTRK gene is a NTRK1 gene,and the one or more point mutations in the NTRK1 gene results in thetranslation of a TrkA protein having substitutions are one or more ofthe following amino acid positions: 33, 336, 337, 324, 420, 444, 517,538, 649, 682, 683, 702, and
 1879. 27. The method of claim 26, whereinthe one or more point mutations in the NTRK1 gene results in thetranslation of a TrkA protein having one or more of the following aminoacid substitutions: R33W, A336E, A337T, R324Q, R324W, V420M, R444Q,R444W, G517R, G517V, K538A, R649W, R649L, R682S, V683G, R702C, andC1879T.
 28. A process for the preparation of crystalline form (I-HS)according to claim 1, comprising: (a) adding concentrated sulfuric acidto a solution of(S)—N-(5-((R)-2-(2,5-difuorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidein EtOH to form the hydrogen sulfate salt of(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamide;(b) adding heptane to the solution in Step (a) to form a slurry; (c)filtering the slurry to isolate(S)—N-(5((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehydrogen sulfate; (d) mixing said(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehydrogen sulfate with a 5:95 w/w solution of water/2-butanone; (e)heating the mixture from step (d) at about 65-70° C. with stirring untilthe weight percent of ethanol is about 0.5% to form a slurry of thecrystalline form of(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehydrogen sulfate; and (f) isolating the crystalline form of(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehydrogen sulfate by filtration.
 29. The method of claim 28, furthercomprising: (b1) seeding the solution from step (a) with(S)—N-(5-((R)-2-(2,5-difluorophenyl)pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidine-1-carboxamidehydrogen sulfate at room temperature and allowing the solution to stiruntil a slurry forms.